DSP-4 dual-specificity phosphatase

ABSTRACT

Compositions and methods are provided for the treatment of conditions associated with cell proliferation, cell differentiation and cell survival. In particular, the dual-specificity phosphatase DSP-4, and polypeptide variants thereof that stimulate dephosphorylation of DSP-4 substrates, are provided. The polypeptides may be used, for example, to identify antibodies and other agents that inhibit DSP-4 activity. The polypeptides and agents may be used to modulate cell proliferation, differentiation and survival.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/544,517, filed Apr. 6, 2000, now allowed, which claims thebenefit of U.S. Provisional Patent Application No. 60/128,204, filedApr. 7, 1999, where are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

[0002] The present invention relates generally to compositions andmethods useful for treating conditions associated with defects in cellproliferation, cell differentiation and/or cell survival. The inventionis more particularly related to dual-specificity protein phosphatases,and polypeptide variants thereof. The present invention is also relatedto the use of such polypeptides to identify antibodies and other agents,including small molecules, that modulate signal transduction leading toproliferative responses, cell differentiation and/or cell survival.

BACKGROUND OF THE INVENTION

[0003] Mitogen-activated protein kinases (MAP-kinases) are present ascomponents of conserved cellular signal transduction pathways that havea variety of conserved members. MAP-kinases are activated byphosphorylation at a dual phosphorylation motif with the sequenceThr-X-Tyr (by MAP-kinase kinases), in which phosphorylation at thetyrosine and threonine residues is required for activity. ActivatedMAP-kinases phosphorylate several transduction targets, includingtranscription factors. Inactivation of MAP-kinases is mediated bydephosphorylation at this site by dual-specificity phosphatases referredto as MAP-kinase phosphatases. In higher eukaryotes, the physiologicalrole of MAP-kinase signaling has been correlated with cellular eventssuch as proliferation, oncogenesis, development and differentiation.Accordingly, the ability to regulate signal transduction via thesepathways could lead to the development of treatments and preventivetherapies for human diseases associated with MAP-kinase signaling, suchas cancer.

[0004] Dual-specificity protein tyrosine phosphatases (dual-specificityphosphatases) are phosphatases that dephosphorylate both phosphotyrosineand phosphothreonine/serine residues (Walton et al., Ann. Rev. Biochem.62:101-120, 1993). Several dual-specificity phosphatases that inactivatea MAP-kinase have been identified, including MKP-1 (WO 97/00315; Keyseand Emslie, Nature 59:644-647, 1992), MKP-4, MKP-5, MKP-7, Hb5 (WO97/06245), PACI (Ward et al., Nature 367:651-654, 1994), HVH2 (Guan andButch, J. Biol. Chem. 270:7197-7203, 1995) and PYST1 (Groom et al., EMBOJ. 15:3621-3632, 1996). Expression of certain dual-specificityphosphatases is induced by stress or mitogens, but others appear to beexpressed constitutively in specific cell types. The regulation ofdual-specificity phosphatase expression and activity is critical forcontrol of MAP-kinase mediated cellular functions, including cellproliferation, cell differentiation and cell survival. For example,dual-specificity phosphatases may function as negative regulators ofcell proliferation. It is likely that there are many suchdual-specificity phosphatases, with varying specificity with regard tocell type or activation. However, the regulation of dual specificityphosphatases remains poorly understood and only a relatively smallnumber of dual-specificity phosphatases have been identified.

[0005] Accordingly, there is a need in the art for an improvedunderstanding of MAP-kinase signaling, and the regulation ofdual-specificity phosphatases within MAP-kinase signaling cascades. Anincreased understanding of dual-specificity phosphatase regulation mayfacilitate the development of methods for modulating the activity ofproteins involved in MAP-kinase cascades, and for treating conditionsassociated with such cascades. The present invention fulfills theseneeds and further provides other related advantages.

SUMMARY OF THE INVENTION

[0006] Briefly stated, the present invention provides compositions andmethods for identifying agents capable of modulating cellularproliferative responses. In one aspect, the present invention providesisolated DSP-4 polypeptides having the sequence of DSP-4 recited in SEQID NO:2, or a variant thereof that differs in one or more amino aciddeletions, additions, insertions or substitutions at no more than 50% ofthe residues in SEQ ID NO:2, such that the polypeptide retains theability to dephosphorylate an activated MAP-kinase.

[0007] Within further aspects, the present invention provides anisolated polynucleotide that encodes at least ten consecutive aminoacids of a polypeptide having a sequence corresponding to SEQ ID NO:2.In certain embodiments the invention provides an isolated polynucleotidethat encodes at least fifteen consecutive amino acids of a polypeptidehaving a sequence corresponding to SEQ ID NO:2. Certain suchpolynucleotides encode a DSP-4 polypeptide. Still further,polynucleotides may be antisense polynucleotides that comprise at least15 consecutive nucleotides complementary to a portion of a DSP-4polynucleotide and/or that detectably hybridize to the complement of thesequence recited in SEQ ID NO:1 under conditions that include a wash in0.1× SSC and 0.1% SDS at 50° C. for 15 minutes. Also provided areexpression vectors comprising any of the foregoing polynucleotides, andhost cells transformed or transfected with such expression vectors.

[0008] The present invention further provides, within other aspects,methods for producing a DSP-4 polypeptide, comprising the steps of: (a)culturing a host cell as described above under conditions that permitexpression of the DSP-4 polypeptide; and (b) isolating DSP-4 polypeptidefrom the host cell culture.

[0009] Also provided by the present invention are isolated antibodies,and antigen binding fragments thereof, that specifically bind to a DSP-4polypeptide such as a polypeptide having the sequence of SEQ ID NO:2.

[0010] The present invention further provides, within other aspects,pharmaceutical compositions comprising a polypeptide, polynucleotide,antibody or fragment thereof as described above in combination with aphysiologically acceptable carrier.

[0011] Within further aspects, the present invention provides methodsfor detecting DSP-4 expression in a sample, comprising: (a) contacting asample with an antibody or an antigen-binding fragment thereof asdescribed above, under conditions and for a time sufficient to allowformation of an antibody/DSP-4 complex; and (b) detecting the level ofantibody/DSP-4 complex.

[0012] Within still other aspects, the present invention providesmethods for detecting DSP-4 expression in a sample, comprising: (a)contacting a sample with an antisense polynucleotide as described above;and (b) detecting in the sample an amount of DSP-4 polynucleotide thathybridizes to the antisense polynucleotide. The amount of DSP-4polynucleotide that hybridizes to the antisense polynucleotide may bedetermined, for example, using polymerase chain reaction or ahybridization assay.

[0013] The invention also provides DSP-4 polypeptides useful inscreening assays for modulators of enzyme activity and/or substratebinding. Methods are also provided, within other aspects, for screeningfor an agent that modulates DSP-4 activity, comprising the steps of: (a)contacting a candidate agent with a DSP-4 polypeptide as describedabove, under conditions and for a time sufficient to permit interactionbetween the polypeptide and candidate agent; and (b) subsequentlyevaluating the ability of the polypeptide to dephosphorylate a DSP-4substrate, relative to a predetermined ability of the polypeptide todephosphorylate the DSP-4 substrate in the absence of candidate agent.Such methods may be performed in vitro or in a cellular environment(e.g., within an intact cell).

[0014] Within further aspects, methods are provided for screening for anagent that modulates DSP-4 activity, comprising the steps of: (a)contacting a candidate agent with a cell comprising a DSP-4 promoteroperably linked to a polynucleotide encoding a detectable transcript orprotein, under conditions and for a time sufficient to permitinteraction between the promoter and candidate agent; and (b)subsequently evaluating the expression of the polynucleotide, relativeto a predetermined level of expression in the absence of candidateagent.

[0015] Also provided are methods for modulating a proliferative responsein a cell, comprising contacting a cell with an agent that modulatesDSP-4 activity.

[0016] Within further aspects, methods are provided for modulatingdifferentiation of a cell, comprising contacting a cell with an agentthat modulates DSP-4 activity.

[0017] The present invention further provides methods for modulatingcell survival, comprising contacting a cell with an agent that modulatesDSP-4 activity.

[0018] Within related aspects, the present invention provides methodsfor treating a patient afflicted with a disorder associated with DSP-4activity (or treatable by administration of DSP-4), comprisingadministering to a patient a therapeutically effective amount of anagent that modulates DSP-4 activity. Such disorders include cancer,graft-versus-host disease, autoimmune diseases, allergies, metabolicdiseases, abnormal cell growth, abnormal cell proliferation and cellcycle abnormalities.

[0019] Within further aspects, DSP-4 substrate trapping mutantpolypeptides are provided. Such polypeptides differ from the sequencerecited in SEQ ID NO:2 in one or more amino acid deletions, additions,insertions or substitutions at no more than 50% of the residues in SEQID NO:2, such that the polypeptide binds to a substrate with an affinitythat is not substantially diminished relative to DSP-4, and such thatthe ability of the polypeptide to dephosphorylate a substrate is reducedrelative to DSP-4. Within certain specific embodiments, a substratetrapping mutant polypeptide contains a substitution at position 119 orposition 150 of SEQ ID NO:2.

[0020] The present invention further provides, within other aspects,methods for screening a molecule for the ability to interact with DSP-4,comprising the steps of: (a) contacting a candidate molecule with apolypeptide as described above under conditions and for a timesufficient to permit the candidate molecule and polypeptide to interact;and (b) detecting the presence or absence of binding of the candidatemolecule to the polypeptide. The step of detecting may comprise, forexample, an affinity purification step, a yeast two hybrid screen or ascreen of a phage display library.

[0021] These and other aspects of the present invention will becomeapparent upon reference to the following detailed description andattached drawings. All references disclosed herein are herebyincorporated by reference in their entirety as if each was incorporatedindividually.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 presents a cDNA sequence for DSP-4 (SEQ ID NO:1), with thestart and stop codons indicated in bold.

[0023]FIG. 2 presents the predicted amino acid sequence of DSP-4 (SEQ IDNO:2).

[0024]FIG. 3 is a sequence alignment showing sequence similarity betweenDSP-4 and other MAP-kinase phosphatases (SEQ ID NOS: 14-21).

[0025]FIG. 4 shows northern blot hybridization using a ³²P-labeled fulllength DSP-4 encoding nucleic acid sequence as probe. Blot containedhuman polyA+RNA from various tissue types as follows: (FIG. 4A) Lane 1,heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver;lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas; (FIG. 4B)Lane 1, spleen; lane 2, thymus; lane 3, prostate; lane 4, testis; lane5, ovary; lane 6, small intestine; lane 7, colon; lane 8, peripheralblood leukocyte.

DETAILED DESCRIPTION OF THE INVENTION

[0026] As noted above, the present invention is generally directed tocompositions and methods for modulating (i.e., stimulating orinhibiting) cellular proliferative responses, in vitro and in vivo. Inparticular, the present invention provides a dual-specificityphosphatase DSP-4 (FIGS. 1-2; SEQ ID NOs:1-2), as well as variantsthereof and antibodies that specifically bind DSP-4. Also providedherein are methods for using such compounds for screens, detectionassays and related therapeutic uses.

DSP-4 POLYPEPTIDES AND POLYNUCLEOTIDES

[0027] As used herein, the term “DSP-4 polypeptide” refers to apolypeptide that comprises a DSP-4 sequence as provided herein or avariant of such a sequence. Such polypeptides are capable ofdephosphorylating both tyrosine and threonine/serine residues in a DSP-4substrate, with an activity that is not substantially diminishedrelative to that of a full length native DSP-4. DSP-4 substrates includeactivated (i.e., phosphorylated) MAP-kinases. Other substrates may beidentified using substrate trapping mutants, as described herein, andinclude polypeptides having one or more phosphorylated tyrosine,threonine and/or serine residues.

[0028] DSP-4 polypeptide variants within the scope of the presentinvention may contain one or more substitutions, deletions, additionsand/or insertions. For certain DSP-4 variants, the ability of thevariant to dephosphorylate tyrosine and threonine residues within aDSP-4 substrate is not substantially diminished. The ability of such aDSP-4 variant to dephosphorylate tyrosine and threonine residues withina DSP-4 substrate may be enhanced or unchanged, relative to a nativeDSP-4, or may be diminished by less than 50%, and preferably less than20%, relative to native DSP-4. Such variants may be identified using therepresentative assays provided herein.

[0029] Also contemplated by the present invention are modified forms ofDSP-4 in which a specific function is disabled. For example, suchproteins may be constitutively active or inactive, or may displayaltered binding or catalytic properties. Such altered proteins may begenerated using well known techniques, and the altered functionconfirmed using screens such as those provided herein. Certain modifiedDSP-4 polypeptides are known as “substrate trapping mutants.” Suchpolypeptides retain the ability to bind a substrate (i.e., K_(m) is notsubstantially diminished), but display a reduced ability todephosphorylate a substrate (i.e., k_(cat) is reduced, preferably toless than 1 per minute). Further, the stability of the substratetrapping mutant/substrate complex should not be substantiallydiminished, relative to the stability of a DSP-4/substrate complex.Complex stability may be assessed based on the association constant(K_(a)). Determination of K_(m), k_(cat) and K_(a) may be readilyaccomplished using standard techniques known in the art (see, e.g., WO98/04712; Lehninger, Biochemistry, 1975 Worth Publishers, NY) and assaysprovided herein. Substrate trapping mutants may be generated, forexample, by modifying DSP-4 with an amino acid substitution at position119 or position 150 (e.g., by replacing the amino acid aspartate atposition 119 with an alanine residue, or by replacing the cysteine atresidue 150 with a serine). Substrate trapping mutants may be used, forexample, to identify DSP-4 substrates. Briefly, the modified DSP-4 maybe contacted with a candidate substrate (alone or within a mixture ofproteins, such as a cell extract) to permit the formation of asubstrate/DSP-4 complex. The complex may then be isolated byconventional techniques to permit the isolation and characterization ofsubstrate. The preparation and use of substrate trapping mutants isdescribed, for example, within PCT Publication No. WO 98/04712.

[0030] Preferably, a variant contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes.

[0031] In general, modifications may be more readily made innon-critical regions, which are regions of the native sequence that donot substantially change the activity of DSP-4. Non-critical regions maybe identified by modifying the DSP-4 sequence in a particular region andassaying the ability of the resulting variant in a phosphatase assay, asdescribed herein. Preferred sequence modifications are made so as toretain the active site domain (VHCNAGVSRAAAIV, SEQ ID NO:3). Withincertain preferred embodiments, such modifications affect interactionsbetween DSP-4 and cellular components other than DSP-4 substrates.However, substitutions may also be made in critical regions of thenative protein, provided that the resulting variant substantiallyretains the ability to stimulate substrate dephosphorylation. Withincertain embodiments, a variant contains substitutions, deletions,additions and/or insertions at no more than 50%, preferably no more than25%, of the amino acid residues.

[0032] Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe activity of the polypeptide. In particular, variants may containadditional amino acid sequences at the amino and/or carboxy termini.Such sequences may be used, for example, to facilitate purification ordetection of the polypeptide.

[0033] DSP-4 polypeptides may be prepared using any of a variety of wellknown techniques. Recombinant polypeptides encoded by DNA sequences asdescribed below may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those having ordinary skillin the art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast and higher eukaryotic cells (includingmammalian cells), and forms that differ in glycosylation may begenerated by varying the host cell or post-isolation processing.Supernatants from suitable host/vector systems which secrete recombinantprotein or polypeptide into culture media may be first concentratedusing a commercially available filter. Following concentration, theconcentrate may be applied to a suitable purification matrix such as anaffinity matrix or an ion exchange resin. Finally, one or more reversephase HPLC steps can be employed to further purify a recombinantpolypeptide.

[0034] Portions and other variants having fewer than about 100 aminoacids, and generally fewer than about 50 amino acids, may also begenerated by synthetic procedures, using techniques well known to thosehaving ordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin-Elmer, Inc., Applied BioSystems Division(Foster City, Calif.), and may be operated according to themanufacturer's instructions.

[0035] A “DSP-4 polynucleotide” is any polynucleotide that encodes atleast a portion of a DSP-4 polypeptide or a variant thereof, or that iscomplementary to such a polynucleotide. Preferred polynucleotidescomprise at least 15 consecutive nucleotides, preferably at least 30consecutive nucleotides, that encode a DSP-4 polypeptide or that arecomplementary to such a sequence. Certain polynucleotides encode a DSP-4polypeptide; others may find use as probes, primers or antisenseoligonucleotides, as described below. Polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

[0036] DSP-4 polynucleotides may comprise a native sequence (i.e., anendogenous DSP-4 sequence or a portion or splice variant thereof) or maycomprise a variant of such a sequence. Polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the activity of the encoded polypeptide is notsubstantially diminished, as described above. The effect on the activityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably at least about 80% identity and most preferably at leastabout 90% identity to a polynucleotide sequence that encodes a nativeDSP-4 or a portion thereof. The percent identity may be readilydetermined by comparing sequences using computer algorithms well knownto those having ordinary skill in the art, such as Align or the BLASTalgorithm (Altschul, J. Mol. Biol. 219:555-565, 1991; Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992), which isavailable at the NCBI website(http://www/ncbi.nlm.nih.gov/cgi-bin/BLAST). Default parameters may beused. Certain variants are substantially homologous to a native gene.Such polynucleotide variants are capable of hybridizing under moderatelystringent conditions to a naturally occurring DNA or RNA sequenceencoding a native DSP-4 (or a complementary sequence). Suitablemoderately stringent conditions include, for example, prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-70° C., 5×SSC, for 1-16 hours; followed by washing once or twice at22-65° C. for 20-40 minutes with one or more each of 2×, 0.5× and 0.2×SSC containing 0.05-0.1% SDS. For additional stringency, conditions mayinclude a wash in 0.1× SSC and 0.1% SDS at 50-60° C. for 15-40 minutes.As known to those having ordinary skill in the art, variations instringency of hybridization conditions may be achieved by altering thetime, temperature and/or concentration of the solutions used forprehybridization, hybridization and wash steps, and suitable conditionsmay also depend in part on the particular nucleotide sequences of theprobe used, and of the blotted, proband nucleic acid sample.Accordingly, it will be appreciated that suitably stringent conditionscan be readily selected without undue experimentation where a desiredselectivity of the probe is identified, based on its ability tohybridize to one or more certain proband sequences while not hybridizingto certain other proband sequences.

[0037] It will also be appreciated by those having ordinary skill in theart that, as a result of the degeneracy of the genetic code, there aremany nucleotide sequences that encode a polypeptide as described herein.Some of these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention.

[0038] Polynucleotides may be prepared using any of a variety oftechniques. For example, a polynucleotide may be amplified from cDNAprepared from a suitable cell or tissue type, such as human thymus orskeletal muscle cells. Such polynucleotides may be amplified viapolymerase chain reaction (PCR). For this approach, sequence-specificprimers may be designed based on the sequences provided herein, and maybe purchased or synthesized.

[0039] An amplified portion may be used to isolate a full length genefrom a suitable library (e.g., human thymus or skeletal muscle cDNA)using well known techniques. Within such techniques, a library (cDNA orgenomic) is screened using one or more polynucleotide probes or primerssuitable for amplification. Preferably, a library is size-selected toinclude larger molecules. Random primed libraries may also be preferredfor identifying 5′ and upstream regions of genes. Genomic libraries arepreferred for obtaining introns and extending 5′ sequences.

[0040] For hybridization techniques, a partial sequence may be labeled(e.g., by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library may then be screened byhybridizing filters containing denatured bacterial colonies (or lawnscontaining phage plaques) with the labeled probe (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. Clones may be analyzed to determine the amount of additionalsequence by, for example, PCR using a primer from the partial sequenceand a primer from the vector. Restriction maps and partial sequences maybe generated to identify one or more overlapping clones. A full lengthcDNA molecule can be generated by ligating suitable fragments, usingwell known techniques.

[0041] Alternatively, there are numerous amplification techniques forobtaining a full length coding sequence from a partial cDNA sequence.Within such techniques, amplification is generally performed via PCR.One such technique is known as “rapid amplification of cDNA ends” orRACE. This technique involves the use of an internal primer and anexternal primer, which hybridizes to a polyA region or vector sequence,to identify sequences that are 5′ and 3′ of a known sequence. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwarewell known in the art. Primers are preferably 17-32 nucleotides inlength, have a GC content of at least 40% and anneal to the targetsequence at temperatures of about 54° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

[0042] A cDNA sequence encoding DSP-4 is provided in FIG. 1 (SEQ IDNO:1), and the predicted amino acid sequence is provided in FIG. 2 (SEQID NO:2). The DSP-4 active site VHCNAGVSRAAAIV (SEQ ID NO:3), is encodedby nucleotide bases located at nucleotide positions 148 through 161 ofSEQ ID NO:1. Sequence information immediately adjacent to this site wasused to design 5′ and 3′ RACE reactions with human skeletal muscle cDNAto identify a 651 base pair cDNA that may be expressed by a variety ofcell types, including heart, testis, thymus and skeletal muscle tissues.This cDNA encodes a protein of 217 amino acids that is referred toherein as dual specificity phosphatase-4, or DSP-4. DSP-4 showssignificant homology to other MAP-kinase phosphatases, as shown by thesequence comparison presented in FIG. 3.

[0043] DSP-4 polynucleotide variants may generally be prepared by anymethod known in the art, including, for example, solid phase chemicalsynthesis. Modifications in a polynucleotide sequence may also beintroduced using standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis. Alternatively, RNAmolecules may be generated by in vitro or in vivo transcription of DNAsequences encoding DSP-4, or a portion thereof, provided that the DNA isincorporated into a vector with a suitable RNA polymerase promoter (suchas T7 or SP6). Certain polynucleotides may be used to prepare an encodedpolypeptide, as described herein. In addition, or alternatively, apolynucleotide may be administered to a patient such that the encodedpolypeptide is generated in vivo.

[0044] A polynucleotide that is complementary to at least a portion of acoding sequence (e.g., an antisense polynucleotide or a ribozyme) mayalso be used as a probe or primer, or to modulate gene expression.Identification of oligonucleotides and ribozymes for use as antisenseagents, and DNA encoding genes for their targeted delivery, involvemethods well known in the art. For example, the desirable properties,lengths and other characteristics of such oligonucleotides are wellknown. Antisense oligonucleotides are typically designed to resistdegradation by endogenous nucleolytic enzymes by using such linkages as:phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl,phosphorodithioate, phosphoramidate, phosphate esters, and other suchlinkages (see, e.g., Agrwal et al., Tetrehedron Lett. 28:3539-3542(1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec etal., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. AcidsRes. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989);Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev.Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 14:97-100(1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of GeneExpression, Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jageret al., Biochemistry 27:7237-7246 (1988)).

[0045] Antisense polynucleotides are oligonucleotides that bind in asequence-specific manner to nucleic acids, such as mRNA or DNA. Whenbound to mRNA that has complementary sequences, antisense preventstranslation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053 to Altman etal.; U.S. Pat. No. 5,190,931 to Inouye, U.S. Pat. No. 5,135,917 toBurch; U.S. Pat. No. 5,087,617 to Smith and Clusel et al. (1993) Nucl.Acids Res. 21:3405-3411, which describes dumbbell antisenseoligonucleotides). Triplex molecules refer to single DNA strands thatbind duplex DNA forming a colinear triplex molecule, thereby preventingtranscription (see, e.g., U.S. Pat. No. 5,176,996 to Hogan et al., whichdescribes methods for making synthetic oligonucleotides that bind totarget sites on duplex DNA).

[0046] Particularly useful antisense nucleotides and triplex moleculesare molecules that are complementary to or bind the sense strand of DNAor mRNA that encodes a DSP-4 polypeptide or a protein mediating anyother process related to expression of endogenous DSP-4, such thatinhibition of translation of mRNA encoding the DSP-4 polypeptide iseffected. cDNA constructs that can be transcribed into antisense RNA mayalso be introduced into cells or tissues to facilitate the production ofantisense RNA. Antisense technology can be used to control geneexpression through interference with binding of polymerases,transcription factors or other regulatory molecules (see Gee et al., InHuber and Carr, Molecular and Immunologic Approaches, Futura PublishingCo. (Mt. Kisco, N.Y.; 1994)). Alternatively, an antisense molecule maybe designed to hybridize with a control region of a DSP-4 gene (e.g.,promoter, enhancer or transcription initiation site), and blocktranscription of the gene; or to block translation by inhibiting bindingof a transcript to ribosomes.

[0047] The present invention also contemplates DSP-4-specific ribozymes.A ribozyme is an RNA molecule that specifically cleaves RNA substrates,such as mRNA, resulting in specific inhibition or interference withcellular gene expression. There are at least five known classes ofribozymes involved in the cleavage and/or ligation of RNA chains.Ribozymes can be targeted to any RNA transcript and can catalyticallycleave such transcripts (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat.No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and5,093,246 to Cech et al.). Any DSP-4 mRNA-specific ribozyme, or anucleic acid encoding such a ribozyme, may be delivered to a host cellto effect inhibition of DSP-4 gene expression. Ribozymes may thereforebe delivered to the host cells by DNA encoding the ribozyme linked to aeukaryotic promoter, such as a eukaryotic viral promoter, such that uponintroduction into the nucleus, the ribozyme will be directlytranscribed.

[0048] Any polynucleotide may be further modified to increase stabilityin vivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiester linkages inthe backbone; and/or the inclusion of nontraditional bases such asinosine, queosine and wybutosine, as well as acetyl- methyl-, thio- andother modified forms of adenine, cytidine, guanine, thymine and uridine.

[0049] Nucleotide sequences as described herein may be joined to avariety of other nucleotide sequences using established recombinant DNAtechniques. For example, a polynucleotide may be cloned into any of avariety of cloning vectors, including plasmids, phagemids, lambda phagederivatives and cosmids. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors andsequencing vectors. In general, a suitable vector contains an origin ofreplication functional in at least one organism, convenient restrictionendonuclease sites and one or more selectable markers. Other elementswill depend upon the desired use, and will be apparent to those havingordinary skill in the art.

[0050] Within certain embodiments, polynucleotides may be formulated soas to permit entry into a cell of a mammal, and expression therein. Suchformulations are particularly useful for therapeutic purposes, asdescribed below. Those having ordinary skill in the art will appreciatethat there are many ways to achieve expression of a polynucleotide in atarget cell, and any suitable method may be employed. For example, apolynucleotide may be incorporated into a viral vector using well knowntechniques. A viral vector may additionally transfer or incorporate agene for a selectable marker (to aid in the identification or selectionof transduced cells) and/or a targeting moiety, such as a gene thatencodes a ligand for a receptor on a specific target cell, to render thevector target specific. Targeting may also be accomplished using anantibody, by methods known to those having ordinary skill in the art.

[0051] Other formulations for therapeutic purposes include colloidaldispersion systems, such as macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. A preferredcolloidal system for use as a delivery vehicle in vitro and in vivo is aliposome (i.e., an artificial membrane vesicle). The preparation and useof such systems is well known in the art.

[0052] Within other aspects, a DSP-4 promoter may be isolated usingstandard techniques. The present invention provides nucleic acidmolecules comprising such a promoter sequence or one or more cis- ortrans-acting regulatory elements thereof. Such regulatory elements mayenhance or suppress expression of DSP-4. A 5′ flanking region may begenerated using standard techniques, based on the genomic sequenceprovided herein. If necessary, additional 5′ sequences may be generatedusing PCR-based or other standard methods. The 5′ region may besubcloned and sequenced using standard methods. Primer extension and/orRNase protection analyses may be used to verify the transcriptionalstart site deduced from the cDNA.

[0053] To define the boundary of the promoter region, putative promoterinserts of varying sizes may be subcloned into a heterologous expressionsystem containing a suitable reporter gene without a promoter orenhancer. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase or the Green Fluorescent Protein gene. Suitableexpression systems are well known and may be prepared using well knowntechniques or obtained commercially. Internal deletion constructs may begenerated using unique internal restriction sites or by partialdigestion of non-unique restriction sites. Constructs may then betransfected into cells that display high levels of DSP-4 expression. Ingeneral, the construct with the minimal 5′ flanking region showing thehighest level of expression of reporter gene is identified as thepromoter. Such promoter regions may be linked to a reporter gene andused to evaluate agents for the ability to modulate DSP-4 transcription.

[0054] Once a functional promoter is identified, cis- and trans-actingelements may be located. Cis-acting sequences may generally beidentified based on homology to previously characterized transcriptionalmotifs. Point mutations may then be generated within the identifiedsequences to evaluate the regulatory role of such sequences. Suchmutations may be generated using site-specific mutagenesis techniques ora PCR-based strategy. The altered promoter is then cloned into areporter gene expression vector, as described above, and the effect ofthe mutation on reporter gene expression is evaluated.

[0055] The present invention also contemplates the use of allelicvariants of DSP-4, as well as DSP-4 sequences from other organisms. Suchsequences may generally be identified based upon similarity to thesequences provided herein (e.g., using hybridization techniques) andbased upon the presence of DSP-4 activity, using an assay providedherein.

[0056] In general, polypeptides and polynucleotides as described hereinare isolated. An “isolated” polypeptide or polynucleotide is one that isremoved from its original environment. For example, anaturally-occurring protein is isolated if it is separated from some orall of the coexisting materials in the natural system. Preferably, suchpolypeptides are at least about 90% pure, more preferably at least about95% pure and most preferably at least about 99% pure. A polynucleotideis considered to be isolated if, for example, it is cloned into a vectorthat is not a part of the natural environment.

ASSAYS FOR DETECTING DSP-4 ACTIVITY

[0057] According to the present invention, substrates of DSP-4 mayinclude full length tyrosine phosphorylated proteins and polypeptides aswell as fragments (e.g., portions), derivatives or analogs thereof thatcan be phosphorylated at a tyrosine residue and that may, in certainpreferred embodiments, also be able to undergo phosphorylation at aserine or a threonine residue. Such fragments, derivatives and analogsinclude any naturally occurring or artificially engineered DSP-4substrate polypeptide that retains at least the biological function ofinteracting with a DSP-4 as provided herein, for example by forming acomplex with a DSP-4. A fragment, derivative or analog of a DSP-4substrate polypeptide, including substrates that are fusion proteins,may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue), and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the substrate polypeptide isfused with another compound, such as a compound to increase thehalf-life of the polypeptide (e.g., polyethylene glycol) or a detectablemoiety such as a reporter molecule, or (iv) one in which additionalamino acids are fused to the substrate polypeptide, including aminoacids that are employed for purification of the substrate polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art. In preferredembodiment, a MAP-kinase polypeptide is a substrate for use as providedherein.

[0058] DSP-4 polypeptide variants may be tested for DSP-4 activity usingany suitable assay for MAP-kinase phosphatase activity. Such assays maybe performed in vitro or within a cell-based assay. For example, aMAP-kinase may be obtained in inactive form from Upstate Biotechnology(Lake Placid, N.Y.; catalog number 14-198), for use as a DSP-4 substrateas provided herein. Phosphorylation of the MAP-kinase can be performedusing well known techniques (such as those described by Zheng and Guan,J. Biol. Chem. 268:16116-16119, 1993) using the MAP-kinase kinase MEK-1(available from Upstate Biotechnology; cat. no. 14-206).

[0059] For example, [³²P]-radiolabeled substrate (e.g., MAP-kinase) maybe used for the kinase reaction, resulting in radiolabeled, activatedMAP-kinase. A DSP-4 polypeptide may then be tested for the ability todephosphorylate an activated MAP-kinase by contacting the DSP-4polypeptide with the MAP-kinase under suitable conditions (e.g., Tris,pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 1 mg/mL bovine serum albumin for10 minutes at 30° C.; or as described by Zheng and Guan, J. Biol. Chem.268:16116-16119, 1993). Dephosphorylation of the MAP-kinase may bedetected using any of a variety of assays, such as a coupled kinaseassay (evaluating phosphorylation of a MAP-kinase substrate using anyassay generally known in the art) or directly, based on (I) the loss ofradioactive phosphate groups (e.g., by gel electrophoresis, followed byautoradiography); (2) the shift in electrophoretic mobility followingdephosphorylation; (3) the loss of reactivity with an antibody specificfor phosphotyrosine or phosphothreonine; or (4) a phosphoamino acidanalysis of the MAP-kinase. Certain assays may generally be performed asdescribed by Ward et al., Nature 367:651-654, 1994 or Alessi et al.,Oncogene 8:2015-2020, 1993. In general, contact of 500 pg-50 ng of DSP-4polypeptide with 100 ng-100 μg activated MAP-kinase should result in adetectable dephosphorylation of the MAP-kinase, typically within 20-30minutes. Within certain embodiments, 0.01-10 units/mL (preferably about0.1 units/mL, where a unit is an amount sufficient to dephosphorylate 1nmol substrate per minute) DSP-4 polypeptide may be contacted with0.1-10 μM (preferably about 1 μM) activated MAP-kinase to produce adetectable dephosphorylation of a MAP-kinase. Preferably, a DSP-4polypeptide results in a dephosphorylation of a MAP-kinase or aphosphorylated substrate (such as a tyrosine- and/orserine-phosphorylated peptide) that is at least as great as thedephosphorylation observed in the presence of a comparable amount ofnative human DSP-4. It will be apparent that other substrates identifiedusing a substrate trapping mutant as described herein may be substitutedfor the MAP-kinase within such assays.

ANTIBODIES AND ANTIGEN-BINDING FRAGMENTS

[0060] Also contemplated by the present invention are peptides,polypeptides, and other non-peptide molecules that specifically bind toa DSP-4. As used herein, a molecule is said to “specifically bind” to aDSP-4 if it reacts at a detectable level with DSP-4, but does not reactdetectably with peptides containing an unrelated sequence, or a sequenceof a different phosphatase. Preferred binding molecules includeantibodies, which may be, for example, polyclonal, monoclonal, singlechain, chimeric, anti-idiotypic, or CDR-grafted immunoglobulins, orfragments thereof, such as proteolytically generated or recombinantlyproduced immunoglobulin F(ab′)₂, Fab, Fv, and Fd fragments. Certainpreferred antibodies are those antibodies that inhibit or block DSP-4activity within an in vitro assay, as described herein. Bindingproperties of an antibody to DSP-4 may generally be assessed usingimmunodetection methods including, for example, an enzyme-linkedimmunosorbent assay (ELISA), immunoprecipitation, immunoblotting and thelike, which may be readily performed by those having ordinary skill inthe art.

[0061] Methods well known in the art may be used to generate antibodies,polyclonal antisera or monoclonal antibodies that are specific for aDSP-4. Antibodies also may be produced as genetically engineeredimmunoglobulins (Ig) or Ig fragments designed to have desirableproperties. For example, by way of illustration and not limitation,antibodies may include a recombinant IgG that is a chimeric fusionprotein having at least one variable (V) region domain from a firstmammalian species and at least one constant region domain from a second,distinct mammalian species. Most commonly, a chimeric antibody hasmurine variable region sequences and human constant region sequences.Such a murine/human chimeric immunoglobulin may be “humanized” bygrafting the complementarity determining regions (CDRs) derived from amurine antibody, which confer binding specificity for an antigen, intohuman-derived V region framework regions and human-derived constantregions. Fragments of these molecules may be generated by proteolyticdigestion, or optionally, by proteolytic digestion followed by mildreduction of disulfide bonds and alkylation. Alternatively, suchfragments may also be generated by recombinant genetic engineeringtechniques.

[0062] As used herein, an antibody is said to be “immunospecific” or to“specifically bind” a DSP-4 polypeptide if it reacts at a detectablelevel with DSP-4, preferably with an affinity constant, K_(a), ofgreater than or equal to about 10⁴ M⁻¹, more preferably of greater thanor equal to about 10⁵ M⁻¹, more preferably of greater than or equal toabout 10⁶ M⁻¹, and still more preferably of greater than or equal toabout 10⁷ M⁻¹. Affinities of binding partners or antibodies can bereadily determined using conventional techniques, for example, thosedescribed by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660 (1949))or by surface plasmon resonance (BIAcore, Biosensor, Piscataway, N.J.).See, e.g., Wolff et al., Cancer Res. 53:2560-2565 (1993).

[0063] Antibodies may generally be prepared by any of a variety oftechniques known to those having ordinary skill in the art. See, e.g.,Harlow et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory (1988). In one such technique, an animal is immunized withDSP-4 as an antigen to generate polyclonal antisera. Suitable animalsinclude, for example, rabbits, sheep, goats, pigs, cattle, and may alsoinclude smaller mammalian species, such as mice, rats, and hamsters, orother species.

[0064] An immunogen may be comprised of cells expressing DSP-4, purifiedor partially purified DSP-4 polypeptides or variants or fragments (e.g.,peptides) thereof, or DSP-4 peptides. DSP-4 peptides may be generated byproteolytic cleavage or may be chemically synthesized. For instance,nucleic acid sequences encoding DSP-4 polypeptides are provided herein,such that those skilled in the art may routinely prepare thesepolypeptides for use as immunogens. Polypeptides or peptides useful forimmunization may also be selected by analyzing the primary, secondary,and tertiary structure of DSP-4 according to methods known to thoseskilled in the art, in order to determine amino acid sequences morelikely to generate an antigenic response in a host animal. See, e.g.,Novotny, 1991 Mol. Immunol. 28:201-207; Berzofsky, 1985 Science229:932-40.

[0065] Preparation of the immunogen for injection into animals mayinclude covalent coupling of the DSP-4 polypeptide (or variant orfragment thereof), to another immunogenic protein, for example, acarrier protein such as keyhole limpet hemocyanin (KLH) or bovine serumalbumin (BSA). In addition, the DSP-4 peptide, polypeptide, orDSP-4-expressing cells to be used as immunogen may be emulsified in anadjuvant. See, e.g., Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988). In general, after the firstinjection, animals receive one or more booster immunizations accordingto a preferred schedule that may vary according to, inter alia, theantigen, the adjuvant (if any) and/or the particular animal species. Theimmune response may be monitored by periodically bleeding the animal,separating the sera out of the collected blood, and analyzing the serain an immunoassay, such as an ELISA or Ouchterlony diffusion assay, orthe like, to determine the specific antibody titer. Once an antibodytiter is established, the animals may be bled periodically to accumulatethe polyclonal antisera. Polyclonal antibodies that bind specifically tothe DSP-4 polypeptide or peptide may then be purified from suchantisera, for example, by affinity chromatography using protein A, orthe DSP-4 polypeptide, immobilized on a suitable solid support.

[0066] Monoclonal antibodies that specifically bind to DSP-4polypeptides or fragments or variants thereof, and hybridomas, which areimmortal eukaryotic cell lines, that produce monoclonal antibodieshaving the desired binding specificity, may also be prepared, forexample, using the technique of Kohler and Milstein (Nature,256:495-497; 1976, Eur. J Immunol. 6:511-519 (1975)) and improvementsthereto. An animal—for example, a rat, hamster, or preferably mouse—isimmunized with a DSP-4 immunogen prepared as described above. Lymphoidcells that include antibody-forming cells, typically spleen cells, areobtained from an immunized animal and may be immortalized by fusion witha drug-sensitized myeloma (e.g., plasmacytoma) cell fusion partner,preferably one that is syngeneic with the immunized animal and thatoptionally has other desirable properties (e.g., inability to expressendogenous Ig gene products). The lymphoid (e.g., spleen) cells and themyeloma cells may be combined for a few minutes with a membranefusion-promoting agent, such as polyethylene glycol or a nonionicdetergent, and then plated at low density on a selective medium thatsupports the growth of hybridoma cells, but not unfused myeloma cells. Apreferred selection media is HAT (hypoxanthine, aminopterin, thymidine).After a sufficient time, usually about one to two weeks, colonies ofcells are observed. Single colonies are isolated, and antibodiesproduced by the cells may be tested for binding activity to the DSP-4polypeptide, or variant or fragment thereof. Hybridomas producingmonoclonal antibodies with high affinity and specificity for a DSP-4antigen are preferred. Hybridomas that produce monoclonal antibodiesthat specifically bind to a DSP-4 polypeptide or variant or fragmentthereof are therefore contemplated by the present invention.

[0067] Monoclonal antibodies may be isolated from the supernatants ofhybridoma cultures. An alternative method for production of a murinemonoclonal antibody is to inject the hybridoma cells into the peritonealcavity of a syngeneic mouse, for example, a mouse that has been treated(e.g., pristane-primed) to promote formation of ascites fluid containingthe monoclonal antibody. Contaminants may be removed from thesubsequently (usually within 1-3 weeks) harvested ascites fluid byconventional techniques, such as chromatography, gel filtration,precipitation, extraction, or the like. For example, antibodies may bepurified by affinity chromatography using an appropriate ligand selectedbased on particular properties of the monoclonal antibody (e.g., heavyor light chain isotype, binding specificity, etc.). Examples of asuitable ligand, immobilized on a solid support, include Protein A,Protein G, an anti-constant region (light chain or heavy chain)antibody, an anti-idiotype antibody and a DSP-4 polypeptide or fragmentor variant thereof.

[0068] Human monoclonal antibodies may be generated by any number oftechniques with which those having ordinary skill in the art will befamiliar. Such methods include but are not limited to, Epstein BarrVirus (EBV) transformation of human peripheral blood cells (e.g.,containing B lymphocytes), in vitro immunization of human B cells,fusion of spleen cells from immunized transgenic mice carrying humanimmunoglobulin genes inserted by yeast artificial chromosomes (YAC),isolation from human immunoglobulin V region phage libraries, or otherprocedures as known in the art and based on the disclosure herein.

[0069] For example, one method for generating human monoclonalantibodies includes immortalizing human peripheral blood cells by EBVtransformation. See, e.g., U.S. Pat. No. 4,464,456. An immortalized cellline producing a monoclonal antibody that specifically binds to a DSP-4polypeptide (or a variant or fragment thereof) can be identified byimmunodetection methods as provided herein, for example, an ELISA, andthen isolated by standard cloning techniques. Another method to generatehuman monoclonal antibodies, in vitro immunization, includes priminghuman splenic B cells with antigen, followed by fusion of primed B cellswith a heterohybrid fusion partner. See, e.g., Boerner et al., 1991 JImmunol. 147:86-95.

[0070] Still another method for the generation of human DSP-4-specificmonoclonal antibodies and polyclonal antisera for use in the presentinvention relates to transgenic mice. See, e.g., U.S. Pat. No.5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58;Jakobovits et al., 1995 Ann. N. Y. Acad. Sci. 764:525-35. In these mice,human immunoglobulin heavy and light chain genes have been artificiallyintroduced by genetic engineering in germline configuration, and theendogenous murine immunoglobulin genes have been inactivated. See, e.g.,Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58. For example,human immunoglobulin transgenes may be mini-gene constructs, ortransloci on yeast artificial chromosomes, which undergo B cell-specificDNA rearrangement and hypermutation in the mouse lymphoid tissue. See,Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58. Humanmonoclonal antibodies specifically binding to DSP-4 may be obtained byimmunizing the transgenic animals, fusing spleen cells with myelomacells, selecting and then cloning cells producing antibody, as describedabove. Polyclonal sera containing human antibodies may also be obtainedfrom the blood of the immunized animals.

[0071] Chimeric antibodies, specific for a DSP-4, including humanizedantibodies, may also be generated according to the present invention. Achimeric antibody has at least one constant region domain derived from afirst mammalian species and at least one variable region domain derivedfrom a second, distinct mammalian species. See, e.g., Morrison et al.,1984, Proc. Natl. Acad Sci. USA, 81:6851-55. In preferred embodiments, achimeric antibody may be constructed by cloning the polynucleotidesequence that encodes at least one variable region domain derived from anon-human monoclonal antibody, such as the variable region derived froma murine, rat, or hamster monoclonal antibody, into a vector containinga nucleic acid sequence that encodes at least one human constant region.See, e.g., Shin et al., 1989 Methods Enzymol. 178:459-76; Walls et al.,1993 Nucleic Acids Res. 21:2921-29. By way of example, thepolynucleotide sequence encoding the light chain variable region of amurine monoclonal antibody may be inserted into a vector containing anucleic acid sequence encoding the human kappa light chain constantregion sequence. In a separate vector, the polynucleotide sequenceencoding the heavy chain variable region of the monoclonal antibody maybe cloned in frame with sequences encoding the human IgG1 constantregion. The particular human constant region selected may depend uponthe effector functions desired for the particular antibody (e.g.,complement fixing, binding to a particular Fc receptor, etc.). Anothermethod known in the art for generating chimeric antibodies is homologousrecombination (e.g., U.S. Pat. No. 5,482,856). Preferably, the vectorswill be transfected into eukaryotic cells for stable expression of thechimeric antibody.

[0072] A non-human/human chimeric antibody may be further geneticallyengineered to create a “humanized” antibody. Such a humanized antibodymay comprise a plurality of CDRs derived from an immunoglobulin of anon-human mammalian species, at least one human variable frameworkregion, and at least one human immunoglobulin constant region.Humanization may in certain embodiments provide an antibody that hasdecreased binding affinity for a DSP-4 when compared, for example, witheither a non-human monoclonal antibody from which a DSP-4 bindingvariable region is obtained, or a chimeric antibody having such a Vregion and at least one human C region, as described above. Usefulstrategies for designing humanized antibodies may therefore include, forexample by way of illustration and not limitation, identification ofhuman variable framework regions that are most homologous to thenon-human framework regions of the chimeric antibody. Without wishing tobe bound by theory, such a strategy may increase the likelihood that thehumanized antibody will retain specific binding affinity for a DSP-4,which in some preferred embodiments may be substantially the sameaffinity for a DSP-4 polypeptide or variant or fragment thereof, and incertain other preferred embodiments may be a greater affinity for DSP-4.See, e.g., Jones et al., 1986 Nature 321:522-25; Riechmann et al., 1988Nature 332:323-27. Designing such a humanized antibody may thereforeinclude determining CDR loop conformations and structural determinantsof the non-human variable regions, for example, by computer modeling,and then comparing the CDR loops and determinants to known human CDRloop structures and determinants. See, e.g., Padlan et al., 1995 FASEB9:133-39; Chothia et al., 1989 Nature, 342:377-383. Computer modelingmay also be used to compare human structural templates selected bysequence homology with the non-human variable regions. See, e.g.,Bajorath et al., 1995 Ther. Immunol. 2:95-103; EP-0578515-A3. Ifhumanization of the non-human CDRs results in a decrease in bindingaffinity, computer modeling may aid in identifying specific amino acidresidues that could be changed by site-directed or other mutagenesistechniques to partially, completely or supra-optimally (i.e., increaseto a level greater than that of the non-humanized antibody) restoreaffinity. Those having ordinary skill in the art are familiar with thesetechniques, and will readily appreciate numerous variations andmodifications to such design strategies.

[0073] Within certain embodiments, the use of antigen-binding fragmentsof antibodies may be preferred. Such fragments include Fab fragments orF(ab′)₂ fragments, which may be prepared by proteolytic digestion withpapain or pepsin, respectively. The antigen binding fragments may beseparated from the Fc fragments by affinity chromatography, for example,using immobilized protein A or protein G, or immobilized DSP-4polypeptide, or a suitable variant or fragment thereof. Those havingordinary skill in the art can routinely and without undueexperimentation determine what is a suitable variant or fragment basedon characterization of affinity purified antibodies obtained, forexample, using immunodetection methods as provided herein. Analternative method to generate Fab fragments includes mild reduction ofF(ab′)₂ fragments followed by alkylation. See, e.g., Weir, Handbook ofExperimental Immunology, 1986, Blackwell Scientific, Boston.

[0074] According to certain embodiments, non-human, human, or humanizedheavy chain and light chain variable regions of any of the abovedescribed Ig molecules may be constructed as single chain Fv (sFv)polypeptide fragments (single chain antibodies). See, e.g., Bird et al.,1988 Science 242:423-426; Huston et al., 1988 Proc. Natl. Acad. Sci. USA85:5879-5883. Multi-functional sFv fusion proteins may be generated bylinking a polynucleotide sequence encoding an sFv polypeptide in-framewith at least one polynucleotide sequence encoding any of a variety ofknown effector proteins. These methods are known in the art, and aredisclosed, for example, in EP-B1-0318554, U.S. Pat. No. 5,132,405, U.S.Pat. No. 5,091,513, and U.S. Pat. No.5,476,786. By way of example,effector proteins may include immunoglobulin constant region sequences.See, e.g., Hollenbaugh et al., 1995 J. Immunol. Methods 188:1-7. Otherexamples of effector proteins are enzymes. As a non-limiting example,such an enzyme may provide a biological activity for therapeuticpurposes (see, e.g., Siemers et al., 1997 Bioconjug. Chem. 8:510-19), ormay provide a detectable activity, such as horseradishperoxidase-catalyzed conversion of any of a number of well-knownsubstrates into a detectable product, for diagnostic uses. Still otherexamples of sFv fusion proteins include Ig-toxin fusions, orimmunotoxins, wherein the sFv polypeptide is linked to a toxin. Thosehaving ordinary skill in the art will appreciate that a wide variety ofpolypeptide sequences have been identified that, under appropriateconditions, are toxic to cells. As used herein, a toxin polypeptide forinclusion in an immunoglobulin-toxin fusion protein may be anypolypeptide capable of being introduced to a cell in a manner thatcompromises cell survival, for example, by directly interfering with avital function or by inducing apoptosis. Toxins thus may include, forexample, ribosome-inactivating proteins, such as Pseudomonas aeruginosaexotoxin A, plant gelonin, bryodin from Bryonia dioica, or the like.See, e.g., Thrush et al., 1996 Annu. Rev. Immunol. 14:49-71; Frankel etal., 1996 Cancer Res. 56:926-32. Numerous other toxins, includingchemotherapeutic agents, anti-mitotic agents, antibiotics, inducers ofapoptosis (or “apoptogens”, see, e.g., Green and Reed, 1998, Science281:1309-1312), or the like, are known to those familiar with the art,and the examples provided herein are intended to be illustrative withoutlimiting the scope and spirit of the invention.

[0075] The sFv may, in certain embodiments, be fused to peptide orpolypeptide domains that permit detection of specific binding betweenthe fusion protein and antigen (e.g., a DSP-4). For example, the fusionpolypeptide domain may be an affinity tag polypeptide. Binding of thesFv fusion protein to a binding partner (e.g., a DSP-4) may therefore bedetected using an affinity polypeptide or peptide tag, such as anavidin, streptavidin or a His (e.g., polyhistidine) tag, by any of avariety of techniques with which those skilled in the art will befamiliar. Detection techniques may also include, for example, binding ofan avidin or streptavidin fusion protein to biotin or to a biotinmimetic sequence (see, e.g., Luo et al., 1998 J. Biotechnol. 65:225 andreferences cited therein), direct covalent modification of a fusionprotein with a detectable moiety (e.g., a labeling moiety), non-covalentbinding of the fusion protein to a specific labeled reporter molecule,enzymatic modification of a detectable substrate by a fusion proteinthat includes a portion having enzyme activity, or immobilization(covalent or non-covalent) of the fusion protein on a solid-phasesupport.

[0076] The sFv fusion protein of the present invention, comprising aDSP-4-specific immunoglobulin-derived polypeptide fused to anotherpolypeptide such as an effector peptide having desirable affinityproperties, may therefore include, for example, a fusion protein whereinthe effector peptide is an enzyme such as glutathione-S-transferase. Asanother example, sFv fusion proteins may also comprise a DSP-4-specificIg polypeptide fused to a Staphylococcus aureus protein A polypeptide;protein A encoding nucleic acids and their use in constructing fusionproteins having affinity for immunoglobulin constant regions aredisclosed generally, for example, in U.S. Pat. No. 5,100,788. Otheruseful affinity polypeptides for construction of sFv fusion proteins mayinclude streptavidin fusion proteins, as disclosed, for example, in WO89/03422; U.S. Pat. No. 5,489,528; U.S. Pat. No. 5,672,691; WO 93/24631;U.S. Pat. No. 5,168,049; U.S. Pat. No. 5,272,254 and elsewhere, andavidin fusion proteins (see, e.g., EP 511,747). As provided herein, sFvpolypeptide sequences may be fused to fusion polypeptide sequences,including effector protein sequences, that may include full lengthfusion polypeptides and that may alternatively contain variants orfragments thereof.

[0077] An additional method for selecting antibodies that specificallybind to a DSP-4 polypeptide or variant or fragment thereof is by phagedisplay. See, e.g., Winter et al., 1994 Annul. Rev. Immunol. 12:433-55;Burton et al., 1994 Adv. Immunol. 57:191-280. Human or murineimmunoglobulin variable region gene combinatorial libraries may becreated in phage vectors that can be screened to select Ig fragments(Fab, Fv, sFv, or multimers thereof) that bind specifically to a DSP-4polypeptide or variant or fragment thereof. See, e.g., U.S. Pat. No.5,223,409; Huse et al., 1989 Science 246:1275-81; Kang et al., 1991Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec.Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 andreferences cited therein. For example, a library containing a pluralityof polynucleotide sequences encoding Ig variable region fragments may beinserted into the genome of a filamentous bacteriophage, such as M13 ora variant thereof, in frame with the sequence encoding a phage coatprotein, for instance, gene III or gene VIII of M13, to create an M13fusion protein. A fusion protein may be a fusion of the coat proteinwith the light chain variable region domain and/or with the heavy chainvariable region domain.

[0078] According to certain embodiments, immunoglobulin Fab fragmentsmay also be displayed on the phage particle, as follows. Polynucleotidesequences encoding Ig constant region domains may be inserted into thephage genome in frame with a coat protein. The phage coat fusion proteinmay thus be fused to an Ig light chain or heavy chain fragment (Fd). Forexample, from a human Ig library, the polynucleotide sequence encodingthe human kappa constant region may be inserted into a vector in framewith the sequence encoding at least one of the phage coat proteins.Additionally or alternatively, the polynucleotide sequence encoding thehuman IgG1 CH1 domain may be inserted in frame with the sequenceencoding at least one other of the phage coat proteins. A plurality ofpolynucleotide sequences encoding variable region domains (e.g., derivedfrom a DNA library) may then be inserted into the vector in frame withthe constant region-coat protein fusions, for expression of Fabfragments fused to a bacteriophage coat protein.

[0079] Phage that display an Ig fragment (e.g., an Ig V-region or Fab)that binds to a DSP-4 polypeptide may be selected by mixing the phagelibrary with DSP-4 or a variant or a fragment thereof, or by contactingthe phage library with a DSP-4 polypeptide immobilized on a solid matrixunder conditions and for a time sufficient to allow binding. Unboundphage are removed by a wash, which typically may be a buffer containingsalt (e.g., NaCl) at a low concentration, preferably with less than 100mM NaCl, more preferably with less than 50 mM NaCl, most preferably withless than 10 mM NaCl, or, alternatively, a buffer containing no salt.Specifically bound phage are then eluted with an NaCl-containing buffer,for example, by increasing the salt concentration in a step-wise manner.Typically, phage that bind the DSP-4 with higher affinity will requirehigher salt concentrations to be released. Eluted phage may bepropagated in an appropriate bacterial host, and generally, successiverounds of DSP-4 binding and elution can be repeated to increase theyield of phage expressing DSP-4 specific immunoglobulin. Combinatorialphage libraries may also be used for humanization of non-human variableregions. See, e.g., Rosok et al., 1996 J. Biol. Chem. 271:22611-18;Rader et al., 1998 Proc. Natl. Acad. Sci. USA 95:8910-15. The DNAsequence of the inserted immunoglobulin gene in the phage so selectedmay be determined by standard techniques. See, Sambrook et al., 1989Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press. Theaffinity selected Ig-encoding sequence may then be cloned into anothersuitable vector for expression of the Ig fragment or, optionally, may becloned into a vector containing Ig constant regions, for expression ofwhole immunoglobulin chains.

[0080] Phage display techniques may also be used to select polypeptides,peptides or single chain antibodies that bind to DSP-4. For examples ofsuitable vectors having multicloning sites into which candidate nucleicacid molecules (e.g., DNA) encoding such peptides or antibodies may beinserted, see, e.g., McLafferty et al., Gene 128:29-36, 1993; Scott etal., 1990 Science 249:386-390; Smith et al., 1993 Methods Enzymol.217:228-257; Fisch et al., 1996, Proc. Natl. Acad. Sci. USA 93:7761-66.The inserted DNA molecules may comprise randomly generated sequences, ormay encode variants of a known peptide or polypeptide domain thatspecifically binds to a DSP-4 polypeptide, or variant or fragmentthereof, as provided herein. Generally, the nucleic acid insert encodesa peptide of up to 60 amino acids, more preferably a peptide of 3 to 35amino acids, and still more preferably a peptide of 6 to 20 amino acids.The peptide encoded by the inserted sequence is displayed on the surfaceof the bacteriophage. Phage expressing a binding domain for a DSP-4polypeptide may be selected on the basis of specific binding to animmobilized DSP-4 polypeptide as described above. As provided herein,well-known recombinant genetic techniques may be used to constructfusion proteins containing the fragment thereof. For example, apolypeptide may be generated that comprises a tandem array of two ormore similar or dissimilar affinity selected DSP-4 binding peptidedomains, in order to maximize binding affinity for DSP-4 of theresulting product.

[0081] In certain other embodiments, the invention contemplates DSP-4specific antibodies that are multimeric antibody fragments. Usefulmethodologies are described generally, for example in Hayden et al.1997, Curr Opin. Immunol. 9:201-12; Coloma et al., 1997 Nat. Biotechnol.15:159-63). For example, multimeric antibody fragments may be created byphage techniques to form miniantibodies (U.S. Pat. No. 5,910 573) ordiabodies (Holliger et al., 1997, Cancer Immunol. Immunother.45:128-130). Multimeric fragments may be generated that are multimers ofa DSP-4-specific Fv, or that are bispecific antibodies comprising aDSP-4-specific Fv noncovalently associated with a second Fv having adifferent antigen specificity. See, e.g., Koelemij et al., 1999 J.Immunother. 22:514-24. As another example, a multimeric antibody maycomprise a bispecific antibody having two single chain antibodies or Fabfragments. According to certain related embodiments, a first Ig fragmentmay be specific for a first antigenic determinant on a DSP-4 polypeptide(or variant or fragment thereof), while a second Ig fragment may bespecific for a second antigenic determinant of the DSP-4 polypeptide.Alternatively, in certain other related embodiments, a firstimmunoglobulin fragment may be specific for an antigenic determinant ona DSP-4 polypeptide or variant or fragment thereof, and a secondimmunoglobulin fragment may be specific for an antigenic determinant ona second, distinct (i.e., non-DSP-4) molecule. Also contemplated arebispecific antibodies that specifically bind DSP-4, wherein at least oneantigen-binding domain is present as a fusion protein.

[0082] Introducing amino acid mutations into DSP-4-bindingimmunoglobulin molecules may be useful to increase the specificity oraffinity for DSP-4, or to alter an effector function. Immunoglobulinswith higher affinity for DSP-4 may be generated by site-directedmutagenesis of particular residues. Computer assisted three-dimensionalmolecular modeling may be employed to identify the amino acid residuesto be changed, in order to improve affinity for the DSP-4 polypeptide .See, e.g., Mountain et al., 1992, Biotechnol. Genet. Eng. Rev. 10:1-142.Alternatively, combinatorial libraries of CDRs may be generated in M13phage and screened for immunoglobulin fragments with improved affinity.See, e.g., Glaser et al., 1992, J. Immunol. 149:3903-3913; Barbas etal., 1994 Proc. Natl. Acad. Sci. USA 91:3809-13; U.S. Pat. No.5,792,456).

[0083] Effector functions may also be altered by site-directedmutagenesis. See, e.g., Duncan et al., 1988 Nature 332:563-64; Morgan etal., 1995 Immunology 86:319-24; Eghtedarzedeh-Kondri et al., 1997Biotechniques 23:830-34. For example, mutation of the glycosylation siteon the Fe portion of the immunoglobulin may alter the ability of theimmunoglobulin to fix complement. See, e.g., Wright et al., 1997 TrendsBiotechnol. 15:26-32. Other mutations in the constant region domains mayalter the ability of the immunoglobulin to fix complement, or to effectantibody-dependent cellular cytotoxicity. See, e.g., Duncan et al., 1988Nature 332:563-64; Morgan et al., 1995 Immunology 86:319-24; Sensel etal., 1997 Mol. Immunol. 34:1019-29.

[0084] The nucleic acid molecules encoding an antibody or fragmentthereof that specifically binds DSP-4, as described herein, may bepropagated and expressed according to any of a variety of well-knownprocedures for nucleic acid excision, ligation, transformation andtransfection. Thus, in certain embodiments expression of an antibodyfragment may be preferred in a prokaryotic host, such as Escherichiacoli (see, e.g., Pluckthun et al., 1989 Methods Enzymol. 178:497-515).In certain other embodiments, expression of the antibody or a fragmentthereof may be preferred in a eukaryotic host cell, including yeast(e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichiapastoris), animal cells (including mammalian cells) or plant cells.Examples of suitable animal cells include, but are not limited to,myeloma, COS, CHO, or hybridoma cells. Examples of plant cells includetobacco, corn, soybean, and rice cells. By methods known to those havingordinary skill in the art and based on the present disclosure, a nucleicacid vector may be designed for expressing foreign sequences in aparticular host system, and then polynucleotide sequences encoding theDSP-4 binding antibody (or fragment thereof) may be inserted. Theregulatory elements will vary according to the particular host.

[0085] A DSP-4-binding immunoglobulin (or fragment thereof) as describedherein may contain a detectable moiety or label such as an enzyme,cytotoxic agent or other reporter molecule, including a dye,radionuclide, luminescent group, fluorescent group, or biotin, or thelike. The DSP-4-specific immunoglobulin or fragment thereof may beradiolabeled for diagnostic or therapeutic applications. Techniques forradiolabeling of antibodies are known in the art. See, e.g., Adams 1998In Vivo 12:11-21; Hiltunen 1993 Acta Oncol. 32:831-9. Therapeuticapplications are described in greater detail below and may include useof the DSP-4-binding antibody (or fragment thereof) in conjunction withother therapeutic agents. The antibody or fragment may also beconjugated to a cytotoxic agent as known in the art and provided herein,for example, a toxin, such as a ribosome-inactivating protein, achemotherapeutic agent, an anti-mitotic agent, an antibiotic or thelike.

[0086] The invention also contemplates the generation of anti-idiotypeantibodies that recognize an antibody (or antigen-binding fragmentthereof) that specifically binds to DSP-4 as provided herein, or avariant or fragment thereof. Anti-idiotype antibodies may be generatedas polyclonal antibodies or as monoclonal antibodies by the methodsdescribed herein, using an anti-DSP-4 antibody (or antigen-bindingfragment thereof) as immunogen. Anti-idiotype antibodies or fragmentsthereof may also be generated by any of the recombinant geneticengineering methods described above, or by phage display selection. Ananti-idiotype antibody may react with the antigen binding site of theanti-DSP-4 antibody such that binding of the anti-DSP-4 antibody to aDSP-4 polypeptide is competitively inhibited. Alternatively, ananti-idiotype antibody as provided herein may not competitively inhibitbinding of an anti-DSP-4 antibody to a DSP-4 polypeptide.

[0087] As provided herein and according to methodologies well known inthe art, polyclonal and monoclonal antibodies may be used for theaffinity isolation of DSP-4 polypeptides. See, e.g., Hermanson et al.,Immobilized Affinity Ligand Techniques, Academic Press, Inc. New York,1992. Briefly, an antibody (or antigen-binding fragment thereof) may beimmobilized on a solid support material, which is then contacted with asample comprising the polypeptide of interest (e.g., a DSP-4). Followingseparation from the remainder of the sample, the polypeptide is thenreleased from the immobilized antibody.

METHODS FOR DETECTING DSP-4 EXPRESSION

[0088] Certain aspects of the present invention provide methods thatemploy antibodies raised against DSP-4, or hybridizing polynucleotides,for diagnostic and assay purposes. Certain assays involve using anantibody or other agent to detect the presence or absence of DSP-4, orproteolytic fragments thereof. Alternatively, nucleic acid encodingDSP-4 may be detected, using standard hybridization and/or PCRtechniques. Suitable probes and primers may be designed by those havingordinary skill in the art based on the DSP-4 cDNA sequence providedherein. Assays may generally be performed using any of a variety ofsamples obtained from a biological source, such as eukaryotic cells,bacteria, viruses, extracts prepared from such organisms and fluidsfound within living organisms. Biological samples that may be obtainedfrom a patient include blood samples, biopsy specimens, tissue explants,organ cultures and other tissue or cell preparations. A patient orbiological source may be a human or non-human animal, a primary cellculture or culture adapted cell line including but not limited togenetically engineered cell lines that may contain chromosomallyintegrated or episomal recombinant nucleic acid sequences, immortalizedor immortalizable cell lines, somatic cell hybrid cell lines,differentiated or differentiatable cell lines, transformed cell linesand the like. In certain preferred embodiments the patient or biologicalsource is a human, and in certain preferred embodiments the biologicalsource is a non-human animal that is a mammal, for example, a rodent(e.g., mouse, rat, hamster, etc.), an ungulate (e.g., bovine) or anon-human primate. In certain other preferred embodiments of theinvention, a patient may be suspected of having or being at risk forhaving a disease associated with altered cellular signal transduction,or may be known to be free of a risk for or presence of such as disease.

[0089] To detect DSP-4 protein, the reagent is typically an antibody,which may be prepared as described below. There are a variety of assayformats known to those having ordinary skill in the art for using anantibody to detect a polypeptide in a sample. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. For example, the assay may be performed in a Western blot format,wherein a protein preparation from the biological sample is resolved bygel electrophoresis, transferred to a suitable membrane and allowed toreact with the antibody. The presence of the antibody on the membranemay then be detected using a suitable detection reagent, as describedbelow.

[0090] In another embodiment, the assay involves the use of antibodyimmobilized on a solid support to bind to the target DSP-4 and remove itfrom the remainder of the sample. The bound DSP-4 may then be detectedusing a second antibody or reagent that contains a reporter group.Alternatively, a competitive assay may be utilized, in which a DSP-4polypeptide is labeled with a reporter group and allowed to bind to theimmobilized antibody after incubation of the antibody with the sample.The extent to which components of the sample inhibit the binding of thelabeled polypeptide to the antibody is indicative of the reactivity ofthe sample with the immobilized antibody, and as a result, indicative ofthe level of DSP-4 in the sample.

[0091] The solid support may be any material known to those havingordinary skill in the art to which the antibody may be attached, such asa test well in a microtiter plate, a nitrocellulose filter or anothersuitable membrane. Alternatively, the support may be a bead or disc,such as glass, fiberglass, latex or a plastic such as polystyrene orpolyvinylchloride. The antibody may be immobilized on the solid supportusing a variety of techniques known to those in the art, which are amplydescribed in the patent and scientific literature.

[0092] In certain embodiments, the assay for detection of DSP-4 in asample is a two-antibody sandwich assay. This assay may be performed byfirst contacting an antibody that has been immobilized on a solidsupport, commonly the well of a microtiter plate, with the biologicalsample, such that DSP-4 within the sample is allowed to bind to theimmobilized antibody (a 30 minute incubation time at room temperature isgenerally sufficient). Unbound sample is then removed from theimmobilized DSP-4/antibody complexes and a second antibody (containing areporter group such as an enzyme, dye, radionuclide, luminescent group,fluorescent group or biotin) capable of binding to a different site onthe DSP-4 is added. The amount of second antibody that remains bound tothe solid support is then determined using a method appropriate for thespecific reporter group. For radioactive groups, scintillation countingor autoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.Standards and standard additions may be used to determine the level ofDSP-4 in a sample, using well known techniques.

[0093] In a related aspect of the present invention, kits for detectingDSP-4 and DSP-4 phosphatase activity are provided. Such kits may bedesigned for detecting the level of DSP-4 or nucleic acid encodingDSP-4, or may detect phosphatase activity of DSP-4 in a directphosphatase assay or a coupled phosphatase assay. In general, the kitsof the present invention comprise one or more containers enclosingelements, such as reagents or buffers, to be used in the assay.

[0094] A kit for detecting the level of DSP-4, or nucleic acid encodingDSP-4, typically contains a reagent that binds to the DSP-4 protein, DNAor RNA. To detect nucleic acid encoding DSP-4, the reagent may be anucleic acid probe or a PCR primer. To detect DSP-4 protein, the reagentis typically an antibody. Such kits also contain a reporter groupsuitable for direct or indirect detection of the reagent (i.e., thereporter group may be covalently bound to the reagent or may be bound toa second molecule, such as Protein A, Protein G, immunoglobulin orlectin, which is itself capable of binding to the reagent). Suitablereporter groups include, but are not limited to, enzymes (e.g.,horseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Suchreporter groups may be used to directly or indirectly detect binding ofthe reagent to a sample component using standard methods known to thosehaving ordinary skill in the art.

[0095] Kits for detecting DSP-4 activity typically comprise a DSP-4substrate in combination with a suitable buffer. DSP-4 activity may bespecifically detected by performing an immunoprecipitation step with aDSP-4-specific antibody prior to performing a phosphatase assay asdescribed above. Other reagents for use in detecting dephosphorylationof substrate may also be provided.

[0096] Within certain diagnostic assays, a proliferative disorder may bedetected in a patient or another biological source organism as providedherein based on the presence of an altered DSP-4 or an altered level ofDSP-4 expression. For example, an antibody may distinguish between awild-type DSP-4 and an altered DSP-4 having a variation in amino acidsequence. Such a variation may be indicative of the presence of aproliferative disorder, or of susceptibility to such a disorder.Hybridization and amplification techniques may be similarly used todetect modified DSP-4 sequences.

METHODS FOR IDENTIFYING MODULATORS OF DSP-4 ACTIVITY

[0097] In one aspect of the present invention, DSP-4 polypeptides may beused to identify agents that modulate DSP-4 activity. Such agents mayinhibit or enhance signal transduction via a MAP-kinase cascade, leadingto cell proliferation. An agent that modulates DSP-4 activity may alterexpression and/or stability of DSP-4, DSP-4 protein activity and/or theability of DSP-4 to dephosphorylate a substrate. Agents that may bescreened within such assays include, but are not limited to, antibodiesand antigen-binding fragments thereof, competing substrates or peptidesthat represent, for example, a catalytic site or a dual phosphorylationmotif, antisense polynucleotides and ribozymes that interfere withtranscription and/or translation of DSP-4 and other natural andsynthetic molecules, for example small molecule inhibitors, that bind toand inactivate DSP-4.

[0098] Candidate agents for use in a method of screening for a modulatorof DSP-4 according to the present invention may be provided as“libraries” or collections of compounds, compositions or molecules. Suchmolecules typically include compounds known in the art as “smallmolecules” and having molecular weights less than 10⁵ daltons,preferably less than 10⁴ daltons and still more preferably less than 10³daltons. For example, members of a library of test compounds can beadministered to a plurality of samples, each containing at least oneDSP-4 polypeptide as provided herein, and then assayed for their abilityto enhance or inhibit DSP-4-mediated dephosphorylation of, or bindingto, a substrate. Compounds so identified as capable of influencing DSP-4function (e.g., phosphotyrosine and/or phosphoserine/threoninedephosphorylation) are valuable for therapeutic and/or diagnosticpurposes, since they permit treatment and/or detection of diseasesassociated with DSP-4 activity. Such compounds are also valuable inresearch directed to molecular signaling mechanisms that involve DSP-4,and to refinements in the discovery and development of future DSP-4compounds exhibiting greater specificity.

[0099] Candidate agents further may be provided as members of acombinatorial library, which preferably includes synthetic agentsprepared according to a plurality of predetermined chemical reactionsperformed in a plurality of reaction vessels. For example, variousstarting compounds may be prepared employing one or more of solid-phasesynthesis, recorded random mix methodologies and recorded reaction splittechniques that permit a given constituent to traceably undergo aplurality of permutations and/or combinations of reaction conditions.The resulting products comprise a library that can be screened followedby iterative selection and synthesis procedures, such as a syntheticcombinatorial library of peptides (see e.g., PCT/US91/08694,PCT/US91/04666, which are hereby incorporated by reference in theirentireties) or other compositions that may include small molecules asprovided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. Pat. No.5,798,035, U.S. Pat. No. 5,789,172, U.S. Pat. No. 5,751,629, which arehereby incorporated by reference in their entireties). Those havingordinary skill in the art will appreciate that a diverse assortment ofsuch libraries may be prepared according to established procedures, andtested using DSP-4 according to the present disclosure.

[0100] In certain embodiments, modulating agents may be identified bycombining a candidate agent with a DSP-4 polypeptide or a polynucleotideencoding such a polypeptide, in vitro or in vivo, and evaluating theeffect of the candidate agent on the DSP-4 phosphatase activity using,for example, a representative assay described herein. An increase ordecrease in phosphatase activity can be measured by performing arepresentative assay provided herein in the presence and absence of acandidate agent. Briefly, a candidate agent may be included in a mixtureof active DSP-4 polypeptide and substrate (e.g., a phosphorylatedMAP-kinase), with or without pre-incubation with one or more componentsof the mixture. In general, a suitable amount of antibody or other agentfor use in such an assay ranges from about 0.01 μM to about 100 μM. Theeffect of the agent on DSP-4 activity may then be evaluated byquantifying the loss of phosphate from the substrate, and comparing theloss with that achieved using DSP-4 without the addition of a candidateagent. Alternatively, a coupled kinase assay may be used, in which DSP-4activity is indirectly measured based on MAP-kinase activity.

[0101] Alternatively, a polynucleotide comprising a DSP-4 promoteroperably linked to a DSP-4 coding region or reporter gene may be used toevaluate the effect of a test compound on DSP-4 transcription. Suchassays may be performed in cells that express DSP-4 endogenously (e.g.,human or other mammalian heart, testis, thymus or skeletal muscle cells)or in cells transfected with an expression vector comprising a DSP-4promoter linked to a reporter gene. The effect of a test compound maythen be evaluated by assaying the effect on transcription of DSP-4 orthe reporter using, for example, a Northern blot analysis or a suitablereporter activity assay.

[0102] DSP-4 activity may also be measured in whole cells transfectedwith a reporter gene whose expression is dependent upon the activationof an appropriate substrate. For example, appropriate cells (i.e., cellsthat express DSP-4) may be transfected with a substrate-dependentpromoter linked to a reporter gene. In such a system, expression of thereporter gene (which may be readily detected using methods well known tothose of ordinary skill in the art) depends upon activation ofsubstrate. Dephosphorylation of substrate may be detected based on adecrease in reporter activity. Candidate modulating agents may be addedto such a system, as described above, to evaluate their effect on DSP-4activity.

[0103] The present invention further provides methods for identifying amolecule that interacts with, or binds to, DSP-4. Such a moleculegenerally associates with DSP-4 with an affinity constant (K_(a)) of atleast 10⁴, preferably at least 10⁵, more preferably at least 10⁶, stillmore preferably at least 10⁷ and most preferably at least 10⁸. Affinityconstants may be determined using well known techniques. Methods foridentifying interacting molecules may be used, for example, as initialscreens for modulating agents, or to identify factors that are involvedin the in vivo DSP-4 activity. Techniques for substrate trapping. Forexample using DSP-4 variants or substrate trapping mutants as describedabove, are also contemplated according to certain embodiments providedherein. In addition to standard binding assays, there are many othertechniques that are well known for identifying interacting molecules,including yeast two-hybrid screens, phage display and affinitytechniques. Such techniques may be performed using routine protocols,which are well known to those having ordinary skill in the art (see,e.g., Bartel et al., In Cellular Interactions in Development: APractical Approach, D. A. Harley, ed., Oxford University Press (Oxford,UK), pp. 153-179, 1993). Within these and other techniques, candidateinteracting proteins (e.g., putative DSP-4 substrates) may bephosphorylated prior to assaying for the presence of DSP-4-binding orinteracting proteins.

[0104] Within other aspects, the present invention provides animalmodels in which an animal either does not express a functional DSP-4, orexpresses an altered DSP-4. Such animals may be generated using standardhomologous recombination strategies. Animal models generated in thismanner may be used to study activities of DSP-4 polypeptides andmodulating agents in vivo.

METHODS FOR DEPHOSPHORYLATING A SUBSTRATE

[0105] In another aspect of the present invention, a DSP-4 polypeptidemay be used for dephosphorylating a substrate of DSP-4 as providedherein. In one embodiment, a substrate may be dephosphorylated in vitroby incubating a DSP-4 polypeptide with a substrate in a suitable buffer(e.g., Tris, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 1 mg/mL bovineserum albumin) for 10 minutes at 30° C. Any compound that can bedephosphorylated by DSP-4, such as a MAP-kinase, may be used as asubstrate. In general, the amounts of the reaction components may rangefrom about 50 pg to about 50 ng of DSP-4 polypeptide and from about 10ng to about 10 μg of substrate. Dephosphorylated substrate may then bepurified, for example, by affinity techniques and/or gelelectrophoresis. The extent of substrate dephosphorylation may generallybe monitored by adding [γ-³²P]labeled substrate to a test aliquot, andevaluating the level of substrate dephosphorylation as described herein.

METHODS FOR MODULATING CELLULAR RESPONSES

[0106] Modulating agents may be used to modulate, modify or otherwisealter (e.g., increase or decrease) cellular responses such as cellproliferation, differentiation and survival, in a variety of contexts,both in vivo and in vitro. In general, to so modulate (e.g., increase ordecrease in a statistically significant manner) such a response, a cellis contacted with an agent that modulates DSP-4 activity, underconditions and for a time sufficient to permit modulation of DSP-4activity. Agents that modulate a cellular response may function in anyof a variety of ways. For example, an agent may modulate a pattern ofgene expression (i.e., may enhance or inhibit expression of a family ofgenes or genes that are expressed in a coordinated fashion). A varietyof hybridization and amplification techniques are available forevaluating patterns of gene expression. Alternatively, or in addition,an agent may effect apoptosis or necrosis of the cell, and/or maymodulate the functioning of the cell cycle within the cell. (See, e.g.,Ashkenazi et al., 1998 Science, 281:1305; Thornberry et al., 1998Science 281:1312; Evan et al., 1998 Science 281:1317; Adams et al., 1998Science 281:1322; and references cited therein.)

[0107] Cells treated as described above may exhibit standardcharacteristics of cells having altered proliferation, differentiationor survival properties. In addition, such cells may (but need not)display alterations in other detectable properties, such as contactinhibition of cell growth, anchorage independent growth or alteredintercellular adhesion. Such properties may be readily detected usingtechniques with which those having ordinary skill in the art will befamiliar.

THERAPEUTIC METHODS

[0108] One or more DSP-4 polypeptides, modulating agents (including anyagent that specifically binds a DSP-4, such as an antibody or fragmentthereof as provided herein) and/or polynucleotides encoding suchpolypeptides and/or modulating agents may also be used to modulate DSP-4activity in a patient. As used herein, a “patient” may be any mammal,including a human, and may be afflicted with a condition associated withDSP-4 activity or may be free of detectable disease. Accordingly, thetreatment may be of an existing disease or may be prophylactic.Conditions associated with DSP-4 activity include any disorderassociated with cell proliferation, including cancer, graft-versus-hostdisease (GVHD), autoimmune diseases, allergy or other conditions inwhich immunosuppression may be involved, metabolic diseases, abnormalcell growth or proliferation and cell cycle abnormalities. Certain suchdisorders involve loss of normal MAP-kinase phosphatase activity,leading to uncontrolled cell growth. DSP-4 polypeptides, andpolynucleotides encoding such polypeptides, can be used to amelioratesuch disorders. Activators of DSP-4 may also be used to treat certaindisorders.

[0109] For administration to a patient, one or more polypeptides,polynucleotides and/or modulating agents are generally formulated as apharmaceutical composition. A pharmaceutical composition may be asterile aqueous or non-aqueous solution, suspension or emulsion, whichadditionally comprises a physiologically acceptable carrier (i.e., anon-toxic material that does not interfere with the activity of theactive ingredient). Such compositions may be in the form of a solid,liquid or gas (aerosol). Alternatively, compositions of the presentinvention may be formulated as a lyophilizate or compounds may beencapsulated within liposomes using well known technology.Pharmaceutical compositions within the scope of the present inventionmay also contain other components, which may be biologically active orinactive. Such components include, but are not limited to, buffers(e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,proteins, polypeptides or amino acids such as glycine, antioxidants,chelating agents such as EDTA or glutathione, stabilizers, dyes,flavoring agents, and suspending agents and/or preservatives.

[0110] Any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of the presentinvention. Carriers for therapeutic use are well known, and aredescribed, for example, in Remingtons Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro ed. 1985). In general, the type of carrieris selected based on the mode of administration. Pharmaceuticalcompositions may be formulated for any appropriate manner ofadministration, including, for example, topical, oral, nasal,intrathecal, rectal, vaginal, sublingual or parenteral administration,including subcutaneous, intravenous, intramuscular, intrasternal,intracavemous, intrameatal or intraurethral injection or infusion. Forparenteral administration, the carrier preferably comprises water,saline, alcohol, a fat, a wax or a buffer. For oral administration, anyof the above carriers or a solid carrier, such as mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,kaolin, glycerin, starch dextrins, sodium alginate,carboxymethylcellulose, ethyl cellulose, glucose, sucrose and/ormagnesium carbonate, may be employed.

[0111] A pharmaceutical composition (e.g., for oral administration ordelivery by injection) may be in the form of a liquid (e.g., an elixir,syrup, solution, emulsion or suspension). A liquid pharmaceuticalcomposition may include, for example, one or more of the following:sterile diluents such as water for injection, saline solution,preferably physiological saline, Ringer's solution, isotonic sodiumchloride, fixed oils such as synthetic mono or diglycerides which mayserve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Aparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. The use ofphysiological saline is preferred, and an injectable pharmaceuticalcomposition is preferably sterile.

[0112] The compositions described herein may be formulated for sustainedrelease (i.e., a formulation such as a capsule or sponge that effects aslow release of compound following administration). Such compositionsmay generally be prepared using well known technology and administeredby, for example, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Sustained-release formulationsmay contain an agent dispersed in a carrier matrix and/or containedwithin a reservoir surrounded by a rate controlling membrane. Carriersfor use within such formulations are biocompatible, and may also bebiodegradable; preferably the formulation provides a relatively constantlevel of active component release. The amount of active compoundcontained within a sustained release formulation depends upon the siteof implantation, the rate and expected duration of release and thenature of the condition to be treated or prevented.

[0113] For pharmaceutical compositions comprising a polynucleotideencoding a DSP-4 polypeptide and/or modulating agent (such that thepolypeptide and/or modulating agent is generated in situ), thepolynucleotide may be present within any of a variety of deliverysystems known to those of ordinary skill in the art, including nucleicacid, and bacterial, viral and mammalian expression systems. Techniquesfor incorporating DNA into such expression systems are well known tothose of ordinary skill in the art. The DNA may also be “naked,” asdescribed, for example, in Ulmer et al., Science 259:1745-1749, 1993 andreviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNAmay be increased by coating the DNA onto biodegradable beads, which areefficiently transported into the cells.

[0114] Within a pharmaceutical composition, a DSP-4 polypeptide,polynucleotide or modulating agent may be linked to any of a variety ofcompounds. For example, such an agent may be linked to a targetingmoiety (e.g., a monoclonal or polyclonal antibody, a protein or aliposome) that facilitates the delivery of the agent to the target site.As used herein, a “targeting moiety” may be any substance (such as acompound or cell) that, when linked to an agent enhances the transportof the agent to a target cell or tissue, thereby increasing the localconcentration of the agent. Targeting moieties include antibodies orfragments thereof, receptors, ligands and other molecules that bind tocells of, or in the vicinity of, the target tissue. An antibodytargeting agent may be an intact (whole) molecule, a fragment thereof,or a functional equivalent thereof. Examples of antibody fragments areF(ab′)₂, -Fab′, Fab and F[v] fragments, which may be produced byconventional methods or by genetic or protein engineering. Linkage isgenerally covalent and may be achieved by, for example, directcondensation or other reactions, or by way of bi- or multi-functionallinkers. Targeting moieties may be selected based on the cell(s) ortissue(s) toward which the agent is expected to exert a therapeuticbenefit.

[0115] Pharmaceutical compositions may be administered in a mannerappropriate to the disease to be treated (or prevented). An appropriatedosage and a suitable duration and frequency of administration will bedetermined by such factors as the condition of the patient, the type andseverity of the patient's disease, the particular form of the activeingredient and the method of administration. In general, an appropriatedosage and treatment regimen provides the agent(s) in an amountsufficient to provide therapeutic and/or prophylactic benefit (e.g., animproved clinical outcome, such as more frequent complete or partialremissions, or longer disease-free and/or overall survival). Forprophylactic use, a dose should be sufficient to prevent, delay theonset of or diminish the severity of a disease associated with cellproliferation.

[0116] Optimal dosages may generally be determined using experimentalmodels and/or clinical trials. In general, the amount of polypeptidepresent in a dose, or produced in situ by DNA present in a dose, rangesfrom about 0.01 μg to about 100 μg per kg of host, typically from about0.1 μg to about 10 μg. The use of the minimum dosage that is sufficientto provide effective therapy is usually preferred. Patients maygenerally be monitored for therapeutic or prophylactic effectivenessusing assays suitable for the condition being treated or prevented,which will be familiar to those having ordinary skill in the art.Suitable dose sizes will vary with the size of the patient, but willtypically range from about 10 mL to about 500 mL for 10-60 kg animal.

[0117] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 Cloning and Sequencing cDNA Encoding DSP-4

[0118] This Example illustrates the cloning of a cDNA molecule encodinghuman DSP-4.

[0119] A conserved sequence motif surrounding the active site domain ofdual-specificity phosphatases was identified as follows: Dualspecificity phosphatases belong to the larger family of protein tyrosinephosphatases (PTPs) that share a conserved catalytic domain containing acysteine residue situated N-terminal to a stretch of five variable aminoacids followed by an arginine residue (Fauman et al., Trends In Bioch.Sci. 21:413-417, 1996). DSPs typically contain a PTP active site motifbut lack sequence homology to PTPs in other regions (Jia, Biochem. andCell Biol. 75:17-26, 1997). There is, however, no reported consensussequence that is conserved among DSPs, nor is a consensus regionapparent from examination of the known DSP sequences such as thosereferred to above. To derive a longer consensus DSP amino acid sequencemotif that would be useful for the identification of new DSP familymembers, multiple known human dual-specificity phosphatases sequenceswere aligned and compared. An alignment of eight amino acid sequencesderived from eight human DSPs having MAP-kinase phosphatase activityyielded a conserved homology region consisting of a 23-amino acidpeptide sequence containing the PTP active site signature motif. Thus, acandidate peptide having the sequence:

GRVLVHCQAGISRSGTNILAYLM  SEQ ID NO:4

[0120] was used to search the Expressed Sequence Tag database (Nat.Center for Biol. Information, www.ncbi.nlm.nih.gov/dbEST). The searchemployed an algorithm (tblastn) capable of reverse translation of thecandidate peptide with iterations allowing for genetic code degeneracywithin default parameters. The search results identified the ESTA1031656 as a candidate MAP-kinase phosphatase. The EST did not includea complete coding region of an expressed gene such as a gene encoding aDSP-4 having MAP-kinase phosphatase activity, nor were the sense strandand open reading frame identified.

[0121] To obtain a full length coding region, human thymus cDNA wasscreened in 5′ and 3′ RACE (rapid amplification of cDNA ends) reactionsas described (Frohman et al., Proc. Nat. Acad. Sci. USA 85:8998, 1988;Ohara et al., Proc. Nat. Acad. Sci. USA 86:5673, 1989; Loh et al.,Science 243:217, 1989) using 5′/3′ RACE kits (Boehringer Mannheim,Indianapolis, Ind.) according to the supplier's instructions. Sequenceinformation immediately adjacent to the conserved sequence motif wasused in the 5′ and 3′ RACE reactions with human skeletal muscle cDNA,using the following primers (SEQ ID NOs:5 to 11): DSP4-SP0:5′-GCATAAAAAAGGCAAGAGAAAAAAGGG-3′ SEQ ID NO:5 DSP4-SP1:5′-CACACTTATTGCTTITCTTTFGCCCT C-3′ SEQ ID NO:6 DSP4-SP2:5′-GCAGTGGTAAATGAGGTTTGTTCAG-3′ SEQ ID NO:7 DSP4-SP3:5′-AGCCCTGGAAACGCCTGC-3′ SEQ ID NO:8 DSP4-SP4:5′-CTGAACAAACCTCATTACCAGTGC3′ SEQ ID NO:9 DSP4-SP5:5′-GCAGGCGTTTCCAGGGCT-3′ SEQ ID NO:1O DSP4-SP6:5′-GAGGGCAAAGAAAGCAATAAGTGTG3′ SEQ ID NO:11

[0122] A cDNA (FIG. 1; SEQ ID NO:1) encoding a protein of 217 aminoacids (FIG. 2; SEQ ID NO:2) was identified as DSP-4. This sequence hassignificant homology to other MAP-kinase phosphatases (FIG. 3). Theidentified cDNA contains the 651 base pair coding region, as well asassociated 5′ and 3′ untranslated sequences. The active site domain forDSP-4 was localized to the region encoded by nucleotides beginning atposition 148 of SEQ ID NO: 1.

[0123] Semiquantitative RT-PCR analyses were performed. These analysesshowed significantly higher levels of DSP-4 mRNA in thymus and skeletalmuscle tissues.

[0124] To localize DSP-4 encoding sequences in human genomic DNA,genomic PCR analysis (Research Genetics, Inc., Huntsville, Ala.) wasconducted using standard methodologies with the following DSP-4-specificoligonucleotide mapping primers: DSP4 +:5′-GTCACACTTATFGCTTTCTTTGCCCTC-3′ SEQ ID NO:12 DSP4 −:5′-GCAGGCGTTTCCAGGGCTGC-3′ SEQ ID NO:13

[0125] The DSP-4 sequences localized to human chromosome 2 in the regionbetween D2S324 and D2S117 (for chromosomal localization nomenclature,see, e.g., Gyapay et al., 1994 Nature Genetics 7:246-339).

Example 2 DSP-4 Expression in Human Tissues

[0126] In this example, a DSP-4 encoding nucleic acid sequence is shownto hybridize to human polyA+RNA from various tissue sources. Full lengthDSP-4 encoding cDNA (SEQ ID NO:1) was ³²P-labeled by the random primermethod as described in Ausubel et al. (1998 Current Protocols inMolecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc.,Boston, Mass.) for use as a nucleic acid hybridization probe. The probewas hybridized to blots containing human polyA+RNA derived from multiplehuman tissues, normalized for the amount of detectable β-actin mRNA(FIG. 4, Cat. No. 7759-1; Clontech, Inc., Palo Alto, Calif.). Blotsunderwent prehybridization for 30 min at 68° C. in Express Hyb™ solution(Clontech), and then were hybridized with the labeled probe for 1 hourat 68° C. in Express Hyb™ solution. The blots were next washed for 40min at room temperature in 2× SSC, 0.05% SDS, followed by a second washfor 40 min at 50° C. in 0.1× SSC, 0.1% SDS. Blots were air-dried andthen exposed to Hyperfilm MP™ autoradiographic film (Amersham LifeSciences, Arlington Hts, Ill.) overnight. Results are shown in FIG. 4,in which the human tissue sources for the RNAs were as follows: (FIG.4A) Lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas;(FIG. 4B) Lane 1, spleen; lane 2, thymus; lane 3, prostate; lane 4,testis; lane 5, ovary; lane 6, small intestine; lane 7, colon; lane 8,peripheral blood leukocyte.

[0127] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein for thepurpose of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, thepresent invention is not limited except as by the appended claims.

1 21 1 1087 DNA Homo sapien 1 acattgcatc cctgggataa acggacctggacaactcact ctcttggtct gtggctgctg 60 cggttacctg gatgggcgaa cacctctgaggctggctttg ttacctgggc aataagggac 120 tagcagttca gccgttttct atgcctgctggatttgtttg tatttgttcc cagccactgc 180 tcatgtaatg tactccctta accaggaaattaaagcattc tcccggaata atctcaggaa 240 gcaatgcacc agggtgacaa cgctaactggaaagaaaatt atagaaacat ggaaagatgc 300 cagaattcat gttgtggaag aagtagagccgagcagtggg ggtggttgtg gttatgtgca 360 ggaccttagc tcggacctgc aagttggcgttattaagcca tggttgctcc tagggtcaca 420 agatgctgct catgatttgg atacactgaaaaagaataag gtgactcata ttcttaatgt 480 tgcatatgga gttgaaaatg ctttcctcagtgactttaca tataagagca tttctatatt 540 ggatctgcct gaaaccaaca tcctgtcttattttccagaa tgttttgaat ttattgaaga 600 agcaaaaaga aaagatggag tggttcttgttcattgtaat gcaggcgttt ccagggctgc 660 tgcaattgta ataggtttcc tgatgaattctgaacaaacc tcatttacca gtgctttttc 720 tttggtgaaa aatgcaagac cttccatatgtccaaattct ggcttcatgg agcagcttcg 780 tacatatcaa gagggcaaag aaagcaataagtgtgacaga atacaggaga acagttcatg 840 agttgcattg tagcagacaa tggacaactgtagtttctga attgacttct atagccatct 900 tttccctttt ttggagagta gactagcaaaactccctttt ttctcttgcc ttttttatgc 960 ataaatggag gtcaatctga ttgtcctgacctactgtata agtaaatttc aaatgtcatt 1020 actttctctt tgttattata atgtgtgattaaatgctttt ttaaattgct aagggaaaat 1080 aaaaaaa 1087 2 217 PRT Homo sapien2 Met Tyr Ser Leu Asn Gln Glu Ile Lys Ala Phe Ser Arg Asn Asn Leu 1 5 1015 Arg Lys Gln Cys Thr Arg Val Thr Thr Leu Thr Gly Lys Lys Ile Ile 20 2530 Glu Thr Trp Lys Asp Ala Arg Ile His Val Val Glu Glu Val Glu Pro 35 4045 Ser Ser Gly Gly Gly Cys Gly Tyr Val Gln Asp Leu Ser Ser Asp Leu 50 5560 Gln Val Gly Val Ile Lys Pro Trp Leu Leu Leu Gly Ser Gln Asp Ala 65 7075 80 Ala His Asp Leu Asp Thr Leu Lys Lys Asn Lys Val Thr His Ile Leu 8590 95 Asn Val Ala Tyr Gly Val Glu Asn Ala Phe Leu Ser Asp Phe Thr Tyr100 105 110 Lys Ser Ile Ser Ile Leu Asp Leu Pro Glu Thr Asn Ile Leu SerTyr 115 120 125 Phe Pro Glu Cys Phe Glu Phe Ile Glu Glu Ala Lys Arg LysAsp Gly 130 135 140 Val Val Leu Val His Cys Asn Ala Gly Val Ser Arg AlaAla Ala Ile 145 150 155 160 Val Ile Gly Phe Leu Met Asn Ser Glu Gln ThrSer Phe Thr Ser Ala 165 170 175 Phe Ser Leu Val Lys Asn Ala Arg Pro SerIle Cys Pro Asn Ser Gly 180 185 190 Phe Met Glu Gln Leu Arg Thr Tyr GlnGlu Gly Lys Glu Ser Asn Lys 195 200 205 Cys Asp Arg Ile Gln Glu Asn SerSer 210 215 3 14 PRT Homo sapien 3 Val His Cys Asn Ala Gly Val Ser ArgAla Ala Ala Ile Val 1 5 10 4 23 PRT Homo sapien 4 Gly Arg Val Leu ValHis Cys Gln Ala Gly Ile Ser Arg Ser Gly Thr 1 5 10 15 Asn Ile Leu AlaTyr Leu Met 20 5 27 DNA Homo sapien 5 gcataaaaaa ggcaagagaa aaaaggg 27 625 DNA Homo sapien 6 cacacttatt gctttctttg ccctc 25 7 25 DNA Homo sapien7 gcactggtaa atgaggtttg ttcag 25 8 18 DNA Homo sapien 8 agccctggaaacgcctgc 18 9 25 DNA Homo sapien 9 ctgaacaaac ctcatttacc agtgc 25 10 18DNA Homo sapien 10 gcaggcgttt ccagggct 18 11 25 DNA Homo sapien 11gagggcaaag aaagcaataa gtgtg 25 12 27 DNA Homo sapien 12 gtcacacttattgctttctt tgccctc 27 13 20 DNA Homo sapien 13 gcaggcgttt ccagggctgc 2014 170 PRT Homo sapien 14 Ser Asp Leu Asp Arg Asp Pro Asn Ser Ala ThrAsp Ser Asp Gly Ser 1 5 10 15 Pro Leu Ser Asn Ser Gln Pro Ser Phe ProVal Glu Ile Leu Pro Phe 20 25 30 Leu Tyr Leu Gly Cys Ala Lys Asp Ser ThrAsn Leu Asp Val Leu Glu 35 40 45 Glu Phe Gly Ile Lys Tyr Ile Leu Asn ValThr Pro Asn Leu Pro Asn 50 55 60 Leu Phe Glu Asn Ala Gly Glu Phe Lys TyrLys Gln Ile Pro Ile Ser 65 70 75 80 Asp His Trp Ser Gln Asn Leu Ser GlnPhe Phe Pro Glu Ala Ile Ser 85 90 95 Phe Ile Asp Glu Ala Arg Gly Lys AsnCys Gly Val Leu Val His Cys 100 105 110 Leu Ala Gly Ile Ser Arg Ser ValThr Val Thr Val Ala Tyr Leu Met 115 120 125 Gln Lys Leu Asn Leu Ser MetAsn Asp Ala Tyr Asp Ile Val Lys Met 130 135 140 Lys Lys Ser Asn Ile SerPro Asn Phe Asn Phe Met Gly Gln Leu Leu 145 150 155 160 Asp Phe Glu ArgThr Leu Gly Leu Ser Ser 165 170 15 168 PRT Homo sapien 15 Asp Arg GluLeu Pro Ser Ser Ala Thr Glu Ser Asp Gly Ser Pro Val 1 5 10 15 Pro SerSer Gln Pro Ala Phe Pro Val Gln Ile Leu Pro Tyr Leu Tyr 20 25 30 Leu GlyCys Ala Lys Asp Ser Thr Asn Leu Asp Val Leu Gly Lys Tyr 35 40 45 Gly IleLys Tyr Ile Leu Asn Val Thr Pro Asn Leu Pro Asn Ala Phe 50 55 60 Glu HisGly Gly Glu Phe Thr Tyr Lys Gln Ile Pro Ile Ser Asp His 65 70 75 80 TrpSer Gln Asn Leu Ser Gln Phe Phe Pro Glu Ala Ile Ser Phe Ile 85 90 95 AspGlu Ala Arg Ser Lys Lys Cys Gly Val Leu Val His Cys Leu Ala 100 105 110Gly Ile Ser Arg Ser Val Thr Val Thr Val Ala Tyr Leu Met Gln Lys 115 120125 Met Asn Leu Ser Leu Asn Asp Ala Tyr Asp Phe Val Lys Arg Lys Lys 130135 140 Ser Asn Ile Ser Pro Asn Phe Asn Phe Met Gly Gln Leu Leu Asp Phe145 150 155 160 Glu Arg Thr Leu Gly Leu Ser Ser 165 16 170 PRT Homosapien 16 Gly Leu Cys Glu Gly Lys Pro Ala Ala Leu Leu Pro Met Ser LeuSer 1 5 10 15 Gln Pro Cys Leu Pro Val Pro Ser Val Gly Leu Thr Arg IleLeu Pro 20 25 30 His Leu Tyr Leu Gly Ser Gln Lys Asp Val Leu Asn Lys AspLeu Met 35 40 45 Thr Gln Asn Gly Ile Ser Tyr Val Leu Asn Ala Ser Asn SerCys Pro 50 55 60 Lys Pro Asp Phe Ile Cys Glu Ser Arg Phe Met Arg Val ProIle Asn 65 70 75 80 Asp Asn Tyr Cys Glu Lys Leu Leu Pro Trp Leu Asp LysSer Ile Glu 85 90 95 Phe Ile Asp Lys Ala Lys Leu Ser Ser Cys Gln Val IleVal His Cys 100 105 110 Leu Ala Gly Ile Ser Arg Ser Ala Thr Ile Ala IleAla Tyr Ile Met 115 120 125 Lys Thr Met Gly Met Ser Ser Asp Asp Ala TyrArg Phe Val Lys Asp 130 135 140 Arg Arg Pro Ser Ile Ser Pro Asn Phe AsnPhe Leu Gly Gln Leu Leu 145 150 155 160 Glu Tyr Glu Arg Thr Leu Lys LeuLeu Ala 165 170 17 168 PRT Homo sapien 17 Pro Ala Gln Ala Leu Pro ProAla Gly Ala Glu Asn Ser Asn Ser Asp 1 5 10 15 Pro Arg Val Pro Ile TyrAsp Gln Gly Gly Pro Val Glu Ile Leu Pro 20 25 30 Tyr Leu Tyr Leu Gly SerCys Asn His Ser Ser Asp Leu Gln Gly Leu 35 40 45 Gln Ala Cys Gly Ile ThrAla Val Leu Asn Val Ser Ala Ser Cys Pro 50 55 60 Asn His Phe Glu Gly LeuPhe His Tyr Lys Ser Ile Pro Val Glu Asp 65 70 75 80 Asn Gln Met Val GluIle Ser Ala Trp Phe Gln Glu Ala Ile Ser Phe 85 90 95 Ile Asp Ser Val LysAsn Ser Gly Gly Arg Val Leu Val His Cys Gln 100 105 110 Ala Gly Ile SerArg Ser Ala Thr Ile Cys Leu Ala Tyr Leu Ile Gln 115 120 125 Ser His ArgVal Arg Leu Asp Glu Ala Phe Asp Phe Val Lys Gln Arg 130 135 140 Arg GlyVal Ile Ser Pro Asn Phe Ser Phe Met Gly Gln Leu Leu Gln 145 150 155 160Leu Glu Thr Gln Val Leu Cys His 165 18 169 PRT Homo sapien 18 Pro LeuSer Thr Ser Val Pro Asp Ser Ala Glu Ser Gly Cys Ser Ser 1 5 10 15 CysSer Thr Pro Leu Tyr Asp Gln Gly Gly Pro Val Glu Ile Leu Pro 20 25 30 PheLeu Tyr Leu Gly Ser Ala Tyr His Ala Ser Arg Lys Asp Met Leu 35 40 45 AspAla Leu Gly Ile Thr Ala Leu Ile Asn Val Ser Ala Asn Cys Pro 50 55 60 AsnHis Phe Glu Gly His Tyr Gln Tyr Lys Ser Ile Pro Val Glu Asp 65 70 75 80Asn His Lys Ala Asp Ile Ser Ser Trp Phe Asn Glu Ala Ile Asp Phe 85 90 95Ile Asp Ser Ile Lys Asn Ala Gly Gly Arg Val Phe Val His Cys Gln 100 105110 Ala Gly Ile Ser Arg Ser Ala Thr Ile Cys Leu Ala Tyr Leu Met Arg 115120 125 Thr Asn Arg Val Lys Leu Asp Glu Ala Phe Glu Phe Val Lys Gln Arg130 135 140 Arg Ser Ile Ile Ser Pro Asn Phe Ser Phe Met Gly Gln Leu LeuGln 145 150 155 160 Phe Glu Ser Gln Val Leu Ala Pro His 165 19 169 PRTHomo sapien 19 Pro Val Pro Pro Ser Ala Thr Glu Pro Leu Asp Leu Gly CysSer Ser 1 5 10 15 Cys Gly Thr Pro Leu His Asp Gln Gly Gly Pro Val GluIle Leu Pro 20 25 30 Phe Leu Tyr Leu Gly Ser Ala Tyr His Ala Ala Arg ArgAsp Met Leu 35 40 45 Asp Ala Leu Gly Ile Thr Ala Leu Leu Asn Val Ser SerAsp Cys Pro 50 55 60 Asn His Phe Glu Gly His Tyr Gln Tyr Lys Cys Ile ProVal Glu Asp 65 70 75 80 Asn His Lys Ala Asp Ile Ser Ser Trp Phe Met GluAla Ile Glu Tyr 85 90 95 Ile Asp Ala Val Lys Asp Cys Arg Gly Arg Val LeuVal His Cys Gln 100 105 110 Ala Gly Ile Ser Arg Ser Ala Thr Ile Cys LeuAla Tyr Leu Met Met 115 120 125 Lys Lys Arg Val Arg Leu Glu Glu Ala PheGlu Phe Val Lys Gln Arg 130 135 140 Arg Ser Ile Ile Ser Pro Asn Phe SerPhe Met Gly Gln Leu Leu Gln 145 150 155 160 Phe Glu Ser Gln Val Leu AlaThr Ser 165 20 171 PRT Homo sapien 20 Ser Glu Arg Ala Leu Ile Ser GlnCys Gly Lys Pro Val Val Asn Val 1 5 10 15 Ser Tyr Arg Pro Ala Tyr AspGln Gly Gly Pro Val Glu Ile Leu Pro 20 25 30 Phe Leu Tyr Leu Gly Ser AlaTyr His Ala Ser Lys Cys Glu Phe Leu 35 40 45 Ala Asn Leu His Ile Thr AlaLeu Leu Asn Val Ser Arg Arg Thr Ser 50 55 60 Glu Ala Cys Met Thr His LeuHis Tyr Lys Trp Ile Pro Val Glu Asp 65 70 75 80 Ser His Thr Ala Asp IleSer Ser His Phe Gln Glu Ala Ile Asp Phe 85 90 95 Ile Asp Cys Val Arg GluLys Gly Gly Lys Val Leu Val His Cys Glu 100 105 110 Ala Gly Ile Ser ArgSer Pro Thr Ile Cys Met Ala Tyr Leu Met Lys 115 120 125 Thr Lys Gln PheArg Leu Lys Glu Ala Phe Asp Tyr Ile Lys Gln Arg 130 135 140 Arg Ser MetVal Ser Pro Asn Phe Gly Phe Met Gly Gln Leu Leu Gln 145 150 155 160 TyrGlu Ser Glu Ile Leu Pro Ser Thr Pro Asn 165 170 21 171 PRT Homo sapien21 His Val Val Glu Glu Val Glu Pro Ser Ser Gly Gly Gly Cys Gly Tyr 1 510 15 Val Gln Asp Leu Ser Ser Asp Leu Gln Val Gly Val Ile Lys Pro Trp 2025 30 Leu Leu Leu Gly Ser Gln Asp Ala Ala His Asp Leu Asp Thr Leu Lys 3540 45 Lys Asn Lys Val Thr His Ile Leu Asn Val Ala Tyr Gly Val Glu Asn 5055 60 Ala Phe Leu Ser Asp Phe Thr Tyr Lys Ser Ile Ser Ile Leu Asp Leu 6570 75 80 Pro Glu Thr Asn Ile Leu Ser Tyr Phe Pro Glu Cys Phe Glu Phe Ile85 90 95 Glu Glu Ala Lys Arg Lys Asp Gly Val Val Leu Val His Cys Asn Ala100 105 110 Gly Val Ser Arg Ala Ala Ala Ile Val Ile Gly Phe Leu Met AsnSer 115 120 125 Glu Gln Thr Ser Phe Thr Ser Ala Phe Ser Leu Val Lys AsnAla Arg 130 135 140 Pro Ser Ile Cys Pro Asn Ser Gly Phe Met Glu Gln IleArg Thr Tyr 145 150 155 160 Gln Glu Gly Lys Glu Ser Asn Lys Cys Asp Arg165 170

1. An isolated polypeptide having the sequence of DSP-4 recited in SEQID NO:2, or a variant thereof that differs in one or more amino aciddeletions, additions, insertions or substitutions at no more than 50% ofthe residues in SEQ ID NO:2, such that the polypeptide retains theability to dephosphorylate an activated MAP-kinase.
 2. An isolatedantibody, or antigen binding fragment thereof, that specifically bindsto a DSP-4 polypeptide having the sequence of SEQ ID NO:2.
 3. Anantibody or fragment thereof according to claim 2, wherein the antibodyis a monoclonal antibody.
 4. A pharmaceutical composition comprising anantibody or fragment thereof according to claim 2 in combination with aphysiologically acceptable carrier.
 5. A method for detecting DSP-4expression in a sample, comprising: (a) contacting a sample with anantibody or an antigen-binding fragment thereof according to claim 2,under conditions and for a time sufficient to allow formation of anantibody/DSP-4 complex; and (b) detecting the level of antibody/DSP-4complex, and therefrom detecting the presence of DSP-4 in a sample.
 6. Amethod according to claim 5, wherein the antibody is linked to a supportmaterial.
 7. A method according to claim 5, wherein the antibody islinked to a detectable marker.
 8. A method according to claim 5, whereinthe sample is a biological sample obtained from a patient.
 9. A methodfor screening for an agent that modulates DSP-4 activity, comprising thesteps of: (a) contacting a candidate agent with a polypeptide accordingto claim 1, under conditions and for a time sufficient to permitinteraction between the polypeptide and candidate agent; and (b)subsequently evaluating the ability of the polypeptide todephosphorylate a DSP-4 substrate, relative to a predetermined abilityof the polypeptide to dephosphorylate the DSP-4 substrate in the absenceof candidate agent; and therefrom identifying an agent that modulatesDSP-4 activity.
 10. A method according to claim 9, wherein the DSP-4substrate is a MAP-kinase.
 11. A method according to claim 9, whereinthe candidate agent is a small molecule.
 12. A method according to claim9, wherein the small molecule is present within a combinatorial library.13. A method for screening for an agent that modulates DSP-4 activity,comprising the steps of: (a) contacting a candidate agent with a cellcomprising a DSP-4 promoter operably linked to a polynucleotide encodinga detectable transcript or protein, under conditions and for a timesufficient to permit interaction between the promoter and candidateagent; and (b) subsequently evaluating the expression of thepolynucleotide, relative to a predetermined level of expression in theabsence of candidate agent; and therefrom identifying an agent thatmodulates DSP-4 activity.
 14. A method according to claim 13, whereinthe polynucleotide encodes a DSP-4 polypeptide.
 15. A method accordingto claim 13, wherein the polynucleotide encodes a reporter protein. 16.A method for modulating a proliferative response in a cell, comprisingcontacting a cell with an agent that modulates DSP-4 activity.
 17. Amethod for modulating differentiation of a cell, comprising contacting acell with an agent that modulates DSP-4 activity.
 18. A method formodulating survival of a cell, comprising contacting a cell with anagent that modulates DSP-4 activity.
 19. A method according to any oneof claims 16-18, wherein the agent modulates a pattern of geneexpression.
 20. A method according to any one of claims 16-18, whereinthe cell displays contact inhibition of cell growth.
 21. A methodaccording to any one of claims 16-18, wherein the cell displaysanchorage independent growth.
 22. A method according to any one ofclaims 16-18, wherein the cell displays an altered intercellularadhesion property.
 23. A method according to claim 18, wherein the agentmodulates apoptosis.
 24. A method according to claim 18, wherein theagent modulates the cell cycle.
 25. A method according to claim 15,wherein the cell is present within a patient.
 26. A method for treatinga patient afflicted with a disorder associated with DSP-4 activity,comprising administering to a patient a therapeutically effective amountof an agent that modulates DSP-4 activity.
 27. A method according toclaim 26, wherein the disorder is selected from the group consisting ofDuchenne muscular dystrophy, cancer, graft-versus-host disease,autoimmune diseases, allergies, metabolic diseases, abnormal cellgrowth, abnormal cell proliferation and cell cycle abnormalities.
 28. ADSP-4 substrate trapping mutant polypeptide that differs from thesequence recited in SEQ ID NO:2 in one or more amino acid deletions,additions, insertions or substitutions at no more than 50% of theresidues in SEQ ID NO:2, such that the polypeptide binds to a substratewith an affinity that is not substantially diminished relative to DSP-4,and such that the ability of the polypeptide to dephosphorylate asubstrate is reduced relative to DSP-4.
 29. A substrate trapping mutantpolypeptide according to claim 28, wherein the polypeptide contains asubstitution at position 118 or position 150 of SEQ ID NO:2.
 30. Amethod for screening a molecule for the ability to interact with DSP-4,comprising the steps of: (a) contacting a candidate molecule with apolypeptide according to claim 1 under conditions and for a timesufficient to permit the candidate molecule and polypeptide to interact;and (b) detecting the presence or absence of binding of the candidatemolecule to the polypeptide, and therefrom determining whether thecandidate molecule interacts with DSP-4.
 31. A method according to claim30, wherein the step of detecting comprises an affinity purificationstep.
 32. A method according to claim 30, wherein the step of detectingcomprises a yeast two hybrid screen or a screen of a phage displaylibrary.